<?xml version="1.0" encoding="US-ASCII"?>
<!-- DFIN New ActiveDisclosure (SM) XBRL Linkbase Document - http://www.dfinsolutions.com/ -->
<!-- Creation Date :2023-08-14T16:16:42.3205+00:00 -->
<!-- Copyright (c) 2023 Donnelly Financial Solutions, Inc. All Rights Reserved. -->
<link:linkbase xsi:schemaLocation="http://www.xbrl.org/2003/linkbase http://www.xbrl.org/2003/xbrl-linkbase-2003-12-31.xsd" xmlns:link="http://www.xbrl.org/2003/linkbase" xmlns:xbrldt="http://xbrl.org/2005/xbrldt" xmlns:xlink="http://www.w3.org/1999/xlink" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance">
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/all" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#all" xlink:type="simple"/>
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#dimension-default" xlink:type="simple"/>
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#dimension-domain" xlink:type="simple"/>
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/domain-member" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#domain-member" xlink:type="simple"/>
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#hypercube-dimension" xlink:type="simple"/>
  <link:roleRef roleURI="http://www.sprucebiosciences.com/20230630/taxonomy/role/StatementCondensedStatementOfStockholdersEquityUnaudited" xlink:href="sprb-20230630.xsd#StatementCondensedStatementOfStockholdersEquityUnaudited" xlink:type="simple"/>
  <link:roleRef roleURI="http://www.sprucebiosciences.com/20230630/taxonomy/role/Role_DisclosureOrganizationAndPrincipalActivitiesAdditionalInformationDetails" xlink:href="sprb-20230630.xsd#Role_DisclosureOrganizationAndPrincipalActivitiesAdditionalInformationDetails" xlink:type="simple"/>
  <link:roleRef roleURI="http://www.sprucebiosciences.com/20230630/taxonomy/role/Role_DisclosureFairValueMeasurementsSummaryOfFinancialAssetsMeasuredAtFairValueOnRecurringBasisDetails" xlink:href="sprb-20230630.xsd#Role_DisclosureFairValueMeasurementsSummaryOfFinancialAssetsMeasuredAtFairValueOnRecurringBasisDetails" xlink:type="simple"/>
  <link:roleRef roleURI="http://www.sprucebiosciences.com/20230630/taxonomy/role/DisclosureFairValueMeasurementsSummaryOfInvestmentsInAnUnrealizedLossPositionDetails" xlink:href="sprb-20230630.xsd#DisclosureFairValueMeasurementsSummaryOfInvestmentsInAnUnrealizedLossPositionDetails" xlink:type="simple"/>
  <link:roleRef roleURI="http://www.sprucebiosciences.com/20230630/taxonomy/role/Role_DisclosureFairValueMeasurementsAdditionalInformationDetails" xlink:href="sprb-20230630.xsd#Role_DisclosureFairValueMeasurementsAdditionalInformationDetails" xlink:type="simple"/>
  <link:roleRef roleURI="http://www.sprucebiosciences.com/20230630/taxonomy/role/Role_DisclosureLeasesAdditionalInformationDetails" xlink:href="sprb-20230630.xsd#Role_DisclosureLeasesAdditionalInformationDetails" xlink:type="simple"/>
  <link:roleRef roleURI="http://www.sprucebiosciences.com/20230630/taxonomy/role/Role_DisclosureTermLoanAdditionalInformationDetails" xlink:href="sprb-20230630.xsd#Role_DisclosureTermLoanAdditionalInformationDetails" xlink:type="simple"/>
  <link:roleRef roleURI="http://www.sprucebiosciences.com/20230630/taxonomy/role/DisclosureCollaborationAndLicenseAgreementsAdditionalInformationDetails" xlink:href="sprb-20230630.xsd#DisclosureCollaborationAndLicenseAgreementsAdditionalInformationDetails" xlink:type="simple"/>
  <link:roleRef roleURI="http://www.sprucebiosciences.com/20230630/taxonomy/role/Role_DisclosureCapitalStructureAdditionalInformationDetails" xlink:href="sprb-20230630.xsd#Role_DisclosureCapitalStructureAdditionalInformationDetails" xlink:type="simple"/>
  <link:roleRef roleURI="http://www.sprucebiosciences.com/20230630/taxonomy/role/Role_DisclosureCapitalStructureSummaryOfCommonStockReservedForFutureIssuanceDetails" xlink:href="sprb-20230630.xsd#Role_DisclosureCapitalStructureSummaryOfCommonStockReservedForFutureIssuanceDetails" xlink:type="simple"/>
  <link:roleRef roleURI="http://www.sprucebiosciences.com/20230630/taxonomy/role/DisclosureStockbasedCompensationExpenseAdditionalInformationDetails" xlink:href="sprb-20230630.xsd#DisclosureStockbasedCompensationExpenseAdditionalInformationDetails" xlink:type="simple"/>
  <link:roleRef roleURI="http://www.sprucebiosciences.com/20230630/taxonomy/role/DisclosureStockbasedCompensationExpenseSummaryOfStockbasedCompensationExpenseDetails" xlink:href="sprb-20230630.xsd#DisclosureStockbasedCompensationExpenseSummaryOfStockbasedCompensationExpenseDetails" xlink:type="simple"/>
  <link:roleRef roleURI="http://www.sprucebiosciences.com/20230630/taxonomy/role/Role_DisclosureNetLossPerShareScheduleOfPotentiallyDilutiveSecuritiesNotIncludedInCalculationOfDilutedPerShareDetails" xlink:href="sprb-20230630.xsd#Role_DisclosureNetLossPerShareScheduleOfPotentiallyDilutiveSecuritiesNotIncludedInCalculationOfDilutedPerShareDetails" xlink:type="simple"/>
  <link:definitionLink xlink:type="extended" xlink:role="http://www.sprucebiosciences.com/20230630/taxonomy/role/StatementCondensedStatementOfStockholdersEquityUnaudited">
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_StatementEquityComponentsAxis" xlink:label="us-gaap_StatementEquityComponentsAxis"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_EquityComponentDomain" xlink:label="us-gaap_EquityComponentDomain"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_EquityComponentDomain" xlink:label="us-gaap_EquityComponentDomain_default"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_StockholdersEquity" xlink:label="us-gaap_StockholdersEquity"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_CommonStockMember" xlink:label="us-gaap_CommonStockMember"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_CommonStockSharesOutstanding" xlink:label="us-gaap_CommonStockSharesOutstanding"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_AdditionalPaidInCapitalMember" xlink:label="us-gaap_AdditionalPaidInCapitalMember"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_StockIssuedDuringPeriodValueStockOptionsExercised" xlink:label="us-gaap_StockIssuedDuringPeriodValueStockOptionsExercised"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_AccumulatedOtherComprehensiveIncomeMember" xlink:label="us-gaap_AccumulatedOtherComprehensiveIncomeMember"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_StockIssuedDuringPeriodSharesStockOptionsExercised" xlink:label="us-gaap_StockIssuedDuringPeriodSharesStockOptionsExercised"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_RetainedEarningsMember" xlink:label="us-gaap_RetainedEarningsMember"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_ExerciseOfPreFundedWarrants" xlink:label="sprb_ExerciseOfPreFundedWarrants"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_ExerciseOfPreFundedWarrantsShares" xlink:label="sprb_ExerciseOfPreFundedWarrantsShares"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_StockIssuedDuringPeriodValueEmployeeStockPurchasePlan" xlink:label="us-gaap_StockIssuedDuringPeriodValueEmployeeStockPurchasePlan"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_StockIssuedDuringPeriodSharesEmployeeStockPurchasePlans" xlink:label="us-gaap_StockIssuedDuringPeriodSharesEmployeeStockPurchasePlans"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_RestrictedStockValueSharesIssuedNetOfTaxWithholdings" xlink:label="us-gaap_RestrictedStockValueSharesIssuedNetOfTaxWithholdings"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_RestrictedStockSharesIssuedNetOfSharesForTaxWithholdings" xlink:label="us-gaap_RestrictedStockSharesIssuedNetOfSharesForTaxWithholdings"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_AdjustmentsToAdditionalPaidInCapitalStockIssuedIssuanceCosts" xlink:label="us-gaap_AdjustmentsToAdditionalPaidInCapitalStockIssuedIssuanceCosts"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_StockIssuedDuringPeriodSharesOfCommonStockAndWarrants" xlink:label="sprb_StockIssuedDuringPeriodSharesOfCommonStockAndWarrants"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_StockIssuedDuringPeriodValueOfCommonStockAndWarrants" xlink:label="sprb_StockIssuedDuringPeriodValueOfCommonStockAndWarrants"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_AdjustmentsToAdditionalPaidInCapitalSharebasedCompensationRequisiteServicePeriodRecognitionValue" xlink:label="us-gaap_AdjustmentsToAdditionalPaidInCapitalSharebasedCompensationRequisiteServicePeriodRecognitionValue"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_OtherComprehensiveIncomeUnrealizedHoldingGainLossOnSecuritiesArisingDuringPeriodNetOfTax" xlink:label="us-gaap_OtherComprehensiveIncomeUnrealizedHoldingGainLossOnSecuritiesArisingDuringPeriodNetOfTax"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_NetIncomeLoss" xlink:label="us-gaap_NetIncomeLoss"/>
    <link:definitionArc xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_StatementEquityComponentsAxis" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_StatementEquityComponentsAxis" xlink:to="us-gaap_EquityComponentDomain" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_StatementEquityComponentsAxis" xlink:to="us-gaap_EquityComponentDomain_default" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StockholdersEquity" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EquityComponentDomain" xlink:to="us-gaap_CommonStockMember" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="1" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_CommonStockSharesOutstanding" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="1" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EquityComponentDomain" xlink:to="us-gaap_AdditionalPaidInCapitalMember" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="2" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StockIssuedDuringPeriodValueStockOptionsExercised" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="2" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EquityComponentDomain" xlink:to="us-gaap_AccumulatedOtherComprehensiveIncomeMember" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="3" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StockIssuedDuringPeriodSharesStockOptionsExercised" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="3" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EquityComponentDomain" xlink:to="us-gaap_RetainedEarningsMember" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="4" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="sprb_ExerciseOfPreFundedWarrants" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="5" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="sprb_ExerciseOfPreFundedWarrantsShares" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="6" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StockIssuedDuringPeriodValueEmployeeStockPurchasePlan" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="7" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StockIssuedDuringPeriodSharesEmployeeStockPurchasePlans" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="8" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_RestrictedStockValueSharesIssuedNetOfTaxWithholdings" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="9" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_RestrictedStockSharesIssuedNetOfSharesForTaxWithholdings" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="10" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_AdjustmentsToAdditionalPaidInCapitalStockIssuedIssuanceCosts" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="11" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="sprb_StockIssuedDuringPeriodSharesOfCommonStockAndWarrants" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="12" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="sprb_StockIssuedDuringPeriodValueOfCommonStockAndWarrants" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="13" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_AdjustmentsToAdditionalPaidInCapitalSharebasedCompensationRequisiteServicePeriodRecognitionValue" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="14" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_OtherComprehensiveIncomeUnrealizedHoldingGainLossOnSecuritiesArisingDuringPeriodNetOfTax" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="15" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_NetIncomeLoss" priority="2" use="optional"/>
  </link:definitionLink>
  <link:definitionLink xlink:type="extended" xlink:role="http://www.sprucebiosciences.com/20230630/taxonomy/role/Role_DisclosureOrganizationAndPrincipalActivitiesAdditionalInformationDetails">
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_OrganizationAndPrincipalActivitiesLineItems" xlink:label="sprb_OrganizationAndPrincipalActivitiesLineItems"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_OrganizationAndPrincipalActivitiesTable" xlink:label="sprb_OrganizationAndPrincipalActivitiesTable"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_SubsequentEventTypeDomain" xlink:label="us-gaap_SubsequentEventTypeDomain"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_SubsequentEventMember" xlink:label="us-gaap_SubsequentEventMember"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_RegistrationPaymentArrangementArrangementDomain" xlink:label="us-gaap_RegistrationPaymentArrangementArrangementDomain"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_ShelfRegistrationAndSalesAgreementMember" xlink:label="sprb_ShelfRegistrationAndSalesAgreementMember"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_SubsidiarySaleOfStockAxis" xlink:label="us-gaap_SubsidiarySaleOfStockAxis"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_RegistrationPaymentArrangementByArrangementAxis" xlink:label="us-gaap_RegistrationPaymentArrangementByArrangementAxis"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_RegistrationPaymentArrangementArrangementDomain" xlink:label="us-gaap_RegistrationPaymentArrangementArrangementDomain_default"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_SaleOfStockNameOfTransactionDomain" xlink:label="us-gaap_SaleOfStockNameOfTransactionDomain"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_PrivatePlacementMember" xlink:label="us-gaap_PrivatePlacementMember"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_TypeOfArrangementAxis" xlink:label="us-gaap_TypeOfArrangementAxis"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="us-gaap_ArrangementsAndNonarrangementTransactionsMember"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="us-gaap_ArrangementsAndNonarrangementTransactionsMember_default"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_SaleOfStockNameOfTransactionDomain" xlink:label="us-gaap_SaleOfStockNameOfTransactionDomain_default"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2022/dei-2022.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2022/dei-2022.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2022/dei-2022.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain_default"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_StatementEquityComponentsAxis" xlink:label="us-gaap_StatementEquityComponentsAxis"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_EquityComponentDomain" xlink:label="us-gaap_EquityComponentDomain"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_CollaborationAndLicenseAgreementKakenPharmaceuticalCoLtdMember" xlink:label="sprb_CollaborationAndLicenseAgreementKakenPharmaceuticalCoLtdMember"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_CommonStockMember" xlink:label="us-gaap_CommonStockMember"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_JefferiesLlcMember" xlink:label="sprb_JefferiesLlcMember"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_StockIssuedDuringPeriodSharesNewIssues" xlink:label="us-gaap_StockIssuedDuringPeriodSharesNewIssues"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_SubsequentEventTypeAxis" xlink:label="us-gaap_SubsequentEventTypeAxis"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_SubsequentEventTypeDomain" xlink:label="us-gaap_SubsequentEventTypeDomain_default"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_PreFundedWarrantsMember" xlink:label="sprb_PreFundedWarrantsMember"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_ClassOfWarrantOrRightNumberOfSecuritiesCalledByWarrantsOrRights" xlink:label="us-gaap_ClassOfWarrantOrRightNumberOfSecuritiesCalledByWarrantsOrRights"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_WarrantMember" xlink:label="us-gaap_WarrantMember"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_ProceedsFromIssuanceOfPrivatePlacement" xlink:label="us-gaap_ProceedsFromIssuanceOfPrivatePlacement"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_MaximumAmountOfSaleCoveredInShelfRegistrationStatement" xlink:label="sprb_MaximumAmountOfSaleCoveredInShelfRegistrationStatement"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_RegistrationPaymentArrangementMaximumPotentialConsideration" xlink:label="us-gaap_RegistrationPaymentArrangementMaximumPotentialConsideration"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_UpfrontPaymentReceived" xlink:label="sprb_UpfrontPaymentReceived"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_CashCashEquivalentsAndShortTermInvestments" xlink:label="us-gaap_CashCashEquivalentsAndShortTermInvestments"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_NetIncomeLoss" xlink:label="us-gaap_NetIncomeLoss"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_NetCashProvidedByUsedInOperatingActivitiesContinuingOperations" xlink:label="us-gaap_NetCashProvidedByUsedInOperatingActivitiesContinuingOperations"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_RetainedEarningsAccumulatedDeficit" xlink:label="us-gaap_RetainedEarningsAccumulatedDeficit"/>
    <link:definitionArc xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="sprb_OrganizationAndPrincipalActivitiesLineItems" xlink:to="sprb_OrganizationAndPrincipalActivitiesTable" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SubsequentEventTypeDomain" xlink:to="us-gaap_SubsequentEventMember" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_RegistrationPaymentArrangementArrangementDomain" xlink:to="sprb_ShelfRegistrationAndSalesAgreementMember" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="sprb_OrganizationAndPrincipalActivitiesTable" xlink:to="us-gaap_SubsidiarySaleOfStockAxis" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_RegistrationPaymentArrangementByArrangementAxis" xlink:to="us-gaap_RegistrationPaymentArrangementArrangementDomain" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_RegistrationPaymentArrangementByArrangementAxis" xlink:to="us-gaap_RegistrationPaymentArrangementArrangementDomain_default" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:to="us-gaap_PrivatePlacementMember" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_TypeOfArrangementAxis" xlink:to="us-gaap_ArrangementsAndNonarrangementTransactionsMember" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_TypeOfArrangementAxis" xlink:to="us-gaap_ArrangementsAndNonarrangementTransactionsMember_default" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_SubsidiarySaleOfStockAxis" xlink:to="us-gaap_SaleOfStockNameOfTransactionDomain" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_SubsidiarySaleOfStockAxis" xlink:to="us-gaap_SaleOfStockNameOfTransactionDomain_default" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain_default" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_StatementEquityComponentsAxis" xlink:to="us-gaap_EquityComponentDomain" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:to="sprb_CollaborationAndLicenseAgreementKakenPharmaceuticalCoLtdMember" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EquityComponentDomain" xlink:to="us-gaap_CommonStockMember" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_EntityDomain" xlink:to="sprb_JefferiesLlcMember" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="sprb_OrganizationAndPrincipalActivitiesLineItems" xlink:to="us-gaap_StockIssuedDuringPeriodSharesNewIssues" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_SubsequentEventTypeAxis" xlink:to="us-gaap_SubsequentEventTypeDomain" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_SubsequentEventTypeAxis" xlink:to="us-gaap_SubsequentEventTypeDomain_default" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="1" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="sprb_OrganizationAndPrincipalActivitiesTable" xlink:to="dei_LegalEntityAxis" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="1" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EquityComponentDomain" xlink:to="sprb_PreFundedWarrantsMember" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="1" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="sprb_OrganizationAndPrincipalActivitiesLineItems" xlink:to="us-gaap_ClassOfWarrantOrRightNumberOfSecuritiesCalledByWarrantsOrRights" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="2" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="sprb_OrganizationAndPrincipalActivitiesTable" xlink:to="us-gaap_TypeOfArrangementAxis" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="2" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EquityComponentDomain" xlink:to="us-gaap_WarrantMember" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="2" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="sprb_OrganizationAndPrincipalActivitiesLineItems" xlink:to="us-gaap_ProceedsFromIssuanceOfPrivatePlacement" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="3" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="sprb_OrganizationAndPrincipalActivitiesTable" xlink:to="us-gaap_RegistrationPaymentArrangementByArrangementAxis" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="3" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="sprb_OrganizationAndPrincipalActivitiesLineItems" xlink:to="sprb_MaximumAmountOfSaleCoveredInShelfRegistrationStatement" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="4" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="sprb_OrganizationAndPrincipalActivitiesTable" xlink:to="us-gaap_StatementEquityComponentsAxis" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="4" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="sprb_OrganizationAndPrincipalActivitiesLineItems" xlink:to="us-gaap_RegistrationPaymentArrangementMaximumPotentialConsideration" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="5" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="sprb_OrganizationAndPrincipalActivitiesTable" xlink:to="us-gaap_SubsequentEventTypeAxis" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="5" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="sprb_OrganizationAndPrincipalActivitiesLineItems" xlink:to="sprb_UpfrontPaymentReceived" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="6" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="sprb_OrganizationAndPrincipalActivitiesLineItems" xlink:to="us-gaap_CashCashEquivalentsAndShortTermInvestments" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="7" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="sprb_OrganizationAndPrincipalActivitiesLineItems" xlink:to="us-gaap_NetIncomeLoss" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="8" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="sprb_OrganizationAndPrincipalActivitiesLineItems" xlink:to="us-gaap_NetCashProvidedByUsedInOperatingActivitiesContinuingOperations" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="9" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="sprb_OrganizationAndPrincipalActivitiesLineItems" xlink:to="us-gaap_RetainedEarningsAccumulatedDeficit" priority="2" use="optional"/>
  </link:definitionLink>
  <link:definitionLink xlink:type="extended" xlink:role="http://www.sprucebiosciences.com/20230630/taxonomy/role/Role_DisclosureFairValueMeasurementsSummaryOfFinancialAssetsMeasuredAtFairValueOnRecurringBasisDetails">
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems" xlink:label="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable" xlink:label="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:label="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_ShortTermDebtMember" xlink:label="us-gaap_ShortTermDebtMember"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_FairValueByFairValueHierarchyLevelAxis" xlink:label="us-gaap_FairValueByFairValueHierarchyLevelAxis"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:label="us-gaap_FairValueMeasurementsFairValueHierarchyDomain"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:label="us-gaap_FairValueMeasurementsFairValueHierarchyDomain_default"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_AvailableForSaleDebtSecuritiesAmortizedCostBasis" xlink:label="us-gaap_AvailableForSaleDebtSecuritiesAmortizedCostBasis"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_FairValueByMeasurementFrequencyAxis" xlink:label="us-gaap_FairValueByMeasurementFrequencyAxis"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_FairValueMeasurementFrequencyDomain" xlink:label="us-gaap_FairValueMeasurementFrequencyDomain"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_FairValueMeasurementFrequencyDomain" xlink:label="us-gaap_FairValueMeasurementFrequencyDomain_default"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_FinancialInstrumentAxis" xlink:label="us-gaap_FinancialInstrumentAxis"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:label="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_default"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_FairValueInputsLevel1Member" xlink:label="us-gaap_FairValueInputsLevel1Member"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_FairValueMeasurementsRecurringMember" xlink:label="us-gaap_FairValueMeasurementsRecurringMember"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_MoneyMarketFundMember" xlink:label="sprb_MoneyMarketFundMember"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_AvailableForSaleDebtSecuritiesAccumulatedGrossUnrealizedGainBeforeTax" xlink:label="us-gaap_AvailableForSaleDebtSecuritiesAccumulatedGrossUnrealizedGainBeforeTax"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_FairValueInputsLevel2Member" xlink:label="us-gaap_FairValueInputsLevel2Member"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_CashAndCashEquivalentMember" xlink:label="sprb_CashAndCashEquivalentMember"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_AvailableForSaleDebtSecuritiesAccumulatedGrossUnrealizedLossBeforeTax" xlink:label="us-gaap_AvailableForSaleDebtSecuritiesAccumulatedGrossUnrealizedLossBeforeTax"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_FairValueInputsLevel3Member" xlink:label="us-gaap_FairValueInputsLevel3Member"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_UsGovernmentTreasurySecurityMember" xlink:label="sprb_UsGovernmentTreasurySecurityMember"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_AvailableForSaleSecuritiesDebtSecurities" xlink:label="us-gaap_AvailableForSaleSecuritiesDebtSecurities"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_USGovernmentAgenciesDebtSecuritiesMember" xlink:label="us-gaap_USGovernmentAgenciesDebtSecuritiesMember"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_ShortTermDebtCorporateBondSecuritiesMember" xlink:label="sprb_ShortTermDebtCorporateBondSecuritiesMember"/>
    <link:definitionArc xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems" xlink:to="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:to="us-gaap_ShortTermDebtMember" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_FairValueByFairValueHierarchyLevelAxis" xlink:to="us-gaap_FairValueMeasurementsFairValueHierarchyDomain" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_FairValueByFairValueHierarchyLevelAxis" xlink:to="us-gaap_FairValueMeasurementsFairValueHierarchyDomain_default" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems" xlink:to="us-gaap_AvailableForSaleDebtSecuritiesAmortizedCostBasis" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable" xlink:to="us-gaap_FairValueByMeasurementFrequencyAxis" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_FairValueByMeasurementFrequencyAxis" xlink:to="us-gaap_FairValueMeasurementFrequencyDomain" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_FairValueByMeasurementFrequencyAxis" xlink:to="us-gaap_FairValueMeasurementFrequencyDomain_default" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_FinancialInstrumentAxis" xlink:to="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_FinancialInstrumentAxis" xlink:to="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_default" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:to="us-gaap_FairValueInputsLevel1Member" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueMeasurementFrequencyDomain" xlink:to="us-gaap_FairValueMeasurementsRecurringMember" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="1" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:to="sprb_MoneyMarketFundMember" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="1" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems" xlink:to="us-gaap_AvailableForSaleDebtSecuritiesAccumulatedGrossUnrealizedGainBeforeTax" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="1" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable" xlink:to="us-gaap_FairValueByFairValueHierarchyLevelAxis" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="1" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:to="us-gaap_FairValueInputsLevel2Member" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="2" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:to="sprb_CashAndCashEquivalentMember" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="2" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems" xlink:to="us-gaap_AvailableForSaleDebtSecuritiesAccumulatedGrossUnrealizedLossBeforeTax" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="2" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable" xlink:to="us-gaap_FinancialInstrumentAxis" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="2" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:to="us-gaap_FairValueInputsLevel3Member" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="3" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:to="sprb_UsGovernmentTreasurySecurityMember" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="3" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems" xlink:to="us-gaap_AvailableForSaleSecuritiesDebtSecurities" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="4" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:to="us-gaap_USGovernmentAgenciesDebtSecuritiesMember" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="5" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:to="sprb_ShortTermDebtCorporateBondSecuritiesMember" priority="2" use="optional"/>
  </link:definitionLink>
  <link:definitionLink xlink:type="extended" xlink:role="http://www.sprucebiosciences.com/20230630/taxonomy/role/DisclosureFairValueMeasurementsSummaryOfInvestmentsInAnUnrealizedLossPositionDetails">
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems" xlink:label="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable" xlink:label="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:label="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_UsGovernmentTreasurySecurityMember" xlink:label="sprb_UsGovernmentTreasurySecurityMember"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_DebtSecuritiesAvailableForSaleContinuousUnrealizedLossPositionLessThan12Months" xlink:label="us-gaap_DebtSecuritiesAvailableForSaleContinuousUnrealizedLossPositionLessThan12Months"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_FinancialInstrumentAxis" xlink:label="us-gaap_FinancialInstrumentAxis"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_CorporateDebtSecuritiesMember" xlink:label="us-gaap_CorporateDebtSecuritiesMember"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_DebtSecuritiesAvailableForSaleContinuousUnrealizedLossPositionLessThan12MonthsAccumulatedLoss" xlink:label="us-gaap_DebtSecuritiesAvailableForSaleContinuousUnrealizedLossPositionLessThan12MonthsAccumulatedLoss"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_DebtSecuritiesAvailableForSaleContinuousUnrealizedLossPosition12MonthsOrLonger" xlink:label="us-gaap_DebtSecuritiesAvailableForSaleContinuousUnrealizedLossPosition12MonthsOrLonger"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_DebtSecuritiesAvailableForSaleContinuousUnrealizedLossPosition12MonthsOrLongerAccumulatedLoss" xlink:label="us-gaap_DebtSecuritiesAvailableForSaleContinuousUnrealizedLossPosition12MonthsOrLongerAccumulatedLoss"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_DebtSecuritiesAvailableForSaleUnrealizedLossPosition" xlink:label="us-gaap_DebtSecuritiesAvailableForSaleUnrealizedLossPosition"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_DebtSecuritiesAvailableForSaleUnrealizedLossPositionAccumulatedLoss" xlink:label="us-gaap_DebtSecuritiesAvailableForSaleUnrealizedLossPositionAccumulatedLoss"/>
    <link:definitionArc xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems" xlink:to="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:to="sprb_UsGovernmentTreasurySecurityMember" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems" xlink:to="us-gaap_DebtSecuritiesAvailableForSaleContinuousUnrealizedLossPositionLessThan12Months" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable" xlink:to="us-gaap_FinancialInstrumentAxis" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_FinancialInstrumentAxis" xlink:to="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="1" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:to="us-gaap_CorporateDebtSecuritiesMember" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="1" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems" xlink:to="us-gaap_DebtSecuritiesAvailableForSaleContinuousUnrealizedLossPositionLessThan12MonthsAccumulatedLoss" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="2" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems" xlink:to="us-gaap_DebtSecuritiesAvailableForSaleContinuousUnrealizedLossPosition12MonthsOrLonger" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="3" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems" xlink:to="us-gaap_DebtSecuritiesAvailableForSaleContinuousUnrealizedLossPosition12MonthsOrLongerAccumulatedLoss" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="4" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems" xlink:to="us-gaap_DebtSecuritiesAvailableForSaleUnrealizedLossPosition" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="5" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems" xlink:to="us-gaap_DebtSecuritiesAvailableForSaleUnrealizedLossPositionAccumulatedLoss" priority="2" use="optional"/>
  </link:definitionLink>
  <link:definitionLink xlink:type="extended" xlink:role="http://www.sprucebiosciences.com/20230630/taxonomy/role/Role_DisclosureFairValueMeasurementsAdditionalInformationDetails">
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems" xlink:label="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable" xlink:label="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_AccruedInvestmentIncomeReceivable" xlink:label="us-gaap_AccruedInvestmentIncomeReceivable"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_FairValueByMeasurementFrequencyAxis" xlink:label="us-gaap_FairValueByMeasurementFrequencyAxis"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_FairValueMeasurementFrequencyDomain" xlink:label="us-gaap_FairValueMeasurementFrequencyDomain"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_FairValueMeasurementsRecurringMember" xlink:label="us-gaap_FairValueMeasurementsRecurringMember"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_DebtSecuritiesAvailableForSaleAllowanceForCreditLoss" xlink:label="us-gaap_DebtSecuritiesAvailableForSaleAllowanceForCreditLoss"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_FairValueMeasurementsNonrecurringMember" xlink:label="us-gaap_FairValueMeasurementsNonrecurringMember"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_DebtInstrumentFairValue" xlink:label="us-gaap_DebtInstrumentFairValue"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_DebtSecuritiesAvailableForSaleRealizedGainLoss" xlink:label="us-gaap_DebtSecuritiesAvailableForSaleRealizedGainLoss"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_DebtSecuritiesAvailableForSaleRealizedGainLossExcludingOtherThanTemporaryImpairment" xlink:label="us-gaap_DebtSecuritiesAvailableForSaleRealizedGainLossExcludingOtherThanTemporaryImpairment"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_CashEquivalentsAndShortTermInvestmentsDueTerm" xlink:label="sprb_CashEquivalentsAndShortTermInvestmentsDueTerm"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_WeightedAverageRemainingContractualTermMaturitiesAvailableForSale" xlink:label="sprb_WeightedAverageRemainingContractualTermMaturitiesAvailableForSale"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_LiabilitiesFairValueDisclosure" xlink:label="us-gaap_LiabilitiesFairValueDisclosure"/>
    <link:definitionArc xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems" xlink:to="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems" xlink:to="us-gaap_AccruedInvestmentIncomeReceivable" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable" xlink:to="us-gaap_FairValueByMeasurementFrequencyAxis" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_FairValueByMeasurementFrequencyAxis" xlink:to="us-gaap_FairValueMeasurementFrequencyDomain" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueMeasurementFrequencyDomain" xlink:to="us-gaap_FairValueMeasurementsRecurringMember" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="1" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems" xlink:to="us-gaap_DebtSecuritiesAvailableForSaleAllowanceForCreditLoss" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="1" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueMeasurementFrequencyDomain" xlink:to="us-gaap_FairValueMeasurementsNonrecurringMember" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="2" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems" xlink:to="us-gaap_DebtInstrumentFairValue" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="3" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems" xlink:to="us-gaap_DebtSecuritiesAvailableForSaleRealizedGainLoss" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="4" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems" xlink:to="us-gaap_DebtSecuritiesAvailableForSaleRealizedGainLossExcludingOtherThanTemporaryImpairment" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="5" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems" xlink:to="sprb_CashEquivalentsAndShortTermInvestmentsDueTerm" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="6" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems" xlink:to="sprb_WeightedAverageRemainingContractualTermMaturitiesAvailableForSale" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="7" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems" xlink:to="us-gaap_LiabilitiesFairValueDisclosure" priority="2" use="optional"/>
  </link:definitionLink>
  <link:definitionLink xlink:type="extended" xlink:role="http://www.sprucebiosciences.com/20230630/taxonomy/role/Role_DisclosureLeasesAdditionalInformationDetails">
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_LesseeLeaseDescriptionLineItems" xlink:label="us-gaap_LesseeLeaseDescriptionLineItems"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2022/elts/srt-2022.xsd#srt_StatementGeographicalAxis" xlink:label="srt_StatementGeographicalAxis"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2022/elts/srt-2022.xsd#srt_SegmentGeographicalDomain" xlink:label="srt_SegmentGeographicalDomain"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_SouthSanFranciscoCaliforniaMember" xlink:label="sprb_SouthSanFranciscoCaliforniaMember"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_LandSubjectToGroundLeases" xlink:label="us-gaap_LandSubjectToGroundLeases"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2022/elts/srt-2022.xsd#srt_SegmentGeographicalDomain" xlink:label="srt_SegmentGeographicalDomain_default"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_OperatingLeaseCommencementMonthAndYear" xlink:label="sprb_OperatingLeaseCommencementMonthAndYear"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_OperatingLeaseTerminationMonthAndYear" xlink:label="sprb_OperatingLeaseTerminationMonthAndYear"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_LesseeOperatingLeaseOptionToExtend" xlink:label="us-gaap_LesseeOperatingLeaseOptionToExtend"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_LesseeOperatingLeaseRenewalTerm" xlink:label="us-gaap_LesseeOperatingLeaseRenewalTerm"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_LesseeOperatingLeaseExistenceOfOptionToExtend" xlink:label="us-gaap_LesseeOperatingLeaseExistenceOfOptionToExtend"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_OperatingLeaseCost" xlink:label="us-gaap_OperatingLeaseCost"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_ShortTermLeaseCommitmentAmount" xlink:label="us-gaap_ShortTermLeaseCommitmentAmount"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_LesseeOperatingLeaseExtendedTermOfContract" xlink:label="sprb_LesseeOperatingLeaseExtendedTermOfContract"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_ShortTermLeaseAdditionalCommitments" xlink:label="sprb_ShortTermLeaseAdditionalCommitments"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_LesseeOperatingLeaseTerminationDate" xlink:label="sprb_LesseeOperatingLeaseTerminationDate"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_ShortTermLeaseCost" xlink:label="us-gaap_ShortTermLeaseCost"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_OperatingLeaseWeightedAverageRemainingLeaseTerm1" xlink:label="us-gaap_OperatingLeaseWeightedAverageRemainingLeaseTerm1"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_OperatingLeaseWeightedAverageDiscountRatePercent" xlink:label="us-gaap_OperatingLeaseWeightedAverageDiscountRatePercent"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_OperatingLeasePayments" xlink:label="us-gaap_OperatingLeasePayments"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_OperatingLeaseLiability" xlink:label="us-gaap_OperatingLeaseLiability"/>
    <link:definitionArc xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_LesseeLeaseDescriptionLineItems" xlink:to="us-gaap_StatementTable" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="srt_StatementGeographicalAxis" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_SegmentGeographicalDomain" xlink:to="sprb_SouthSanFranciscoCaliforniaMember" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LesseeLeaseDescriptionLineItems" xlink:to="us-gaap_LandSubjectToGroundLeases" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_StatementGeographicalAxis" xlink:to="srt_SegmentGeographicalDomain" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_StatementGeographicalAxis" xlink:to="srt_SegmentGeographicalDomain_default" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="1" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LesseeLeaseDescriptionLineItems" xlink:to="sprb_OperatingLeaseCommencementMonthAndYear" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="2" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LesseeLeaseDescriptionLineItems" xlink:to="sprb_OperatingLeaseTerminationMonthAndYear" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="3" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LesseeLeaseDescriptionLineItems" xlink:to="us-gaap_LesseeOperatingLeaseOptionToExtend" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="4" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LesseeLeaseDescriptionLineItems" xlink:to="us-gaap_LesseeOperatingLeaseRenewalTerm" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="5" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LesseeLeaseDescriptionLineItems" xlink:to="us-gaap_LesseeOperatingLeaseExistenceOfOptionToExtend" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="6" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LesseeLeaseDescriptionLineItems" xlink:to="us-gaap_OperatingLeaseCost" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="7" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LesseeLeaseDescriptionLineItems" xlink:to="us-gaap_ShortTermLeaseCommitmentAmount" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="8" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LesseeLeaseDescriptionLineItems" xlink:to="sprb_LesseeOperatingLeaseExtendedTermOfContract" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="9" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LesseeLeaseDescriptionLineItems" xlink:to="sprb_ShortTermLeaseAdditionalCommitments" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="10" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LesseeLeaseDescriptionLineItems" xlink:to="sprb_LesseeOperatingLeaseTerminationDate" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="11" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LesseeLeaseDescriptionLineItems" xlink:to="us-gaap_ShortTermLeaseCost" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="12" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LesseeLeaseDescriptionLineItems" xlink:to="us-gaap_OperatingLeaseWeightedAverageRemainingLeaseTerm1" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="13" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LesseeLeaseDescriptionLineItems" xlink:to="us-gaap_OperatingLeaseWeightedAverageDiscountRatePercent" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="14" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LesseeLeaseDescriptionLineItems" xlink:to="us-gaap_OperatingLeasePayments" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="15" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LesseeLeaseDescriptionLineItems" xlink:to="us-gaap_OperatingLeaseLiability" priority="2" use="optional"/>
  </link:definitionLink>
  <link:definitionLink xlink:type="extended" xlink:role="http://www.sprucebiosciences.com/20230630/taxonomy/role/Role_DisclosureTermLoanAdditionalInformationDetails">
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_DebtInstrumentLineItems" xlink:label="us-gaap_DebtInstrumentLineItems"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_DebtInstrumentTable" xlink:label="us-gaap_DebtInstrumentTable"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_DebtInstrumentAxis" xlink:label="us-gaap_DebtInstrumentAxis"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_VariableRateAxis" xlink:label="us-gaap_VariableRateAxis"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_VariableRateDomain" xlink:label="us-gaap_VariableRateDomain"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_VariableRateDomain" xlink:label="us-gaap_VariableRateDomain_default"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_TypeOfArrangementAxis" xlink:label="us-gaap_TypeOfArrangementAxis"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="us-gaap_ArrangementsAndNonarrangementTransactionsMember"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2022/elts/srt-2022.xsd#srt_RangeAxis" xlink:label="srt_RangeAxis"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2022/elts/srt-2022.xsd#srt_RangeMember" xlink:label="srt_RangeMember"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2022/elts/srt-2022.xsd#srt_RangeMember" xlink:label="srt_RangeMember_default"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2022/elts/srt-2022.xsd#srt_MaximumMember" xlink:label="srt_MaximumMember"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2022/dei-2022.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2022/dei-2022.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_ClassOfWarrantOrRightDomain" xlink:label="us-gaap_ClassOfWarrantOrRightDomain"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_WarrantMember" xlink:label="us-gaap_WarrantMember"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_StatementEquityComponentsAxis" xlink:label="us-gaap_StatementEquityComponentsAxis"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_EquityComponentDomain" xlink:label="us-gaap_EquityComponentDomain"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_FirstAmendmentToLoanAndSecurityAgreementMember" xlink:label="sprb_FirstAmendmentToLoanAndSecurityAgreementMember"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_CommonStockMember" xlink:label="us-gaap_CommonStockMember"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_PrimeRateMember" xlink:label="us-gaap_PrimeRateMember"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_DebtInstrumentNameDomain" xlink:label="us-gaap_DebtInstrumentNameDomain"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_DebtInstrumentNameDomain" xlink:label="us-gaap_DebtInstrumentNameDomain_default"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_TermLoanMember" xlink:label="sprb_TermLoanMember"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_ClassOfWarrantOrRightAxis" xlink:label="us-gaap_ClassOfWarrantOrRightAxis"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_ClassOfWarrantOrRightDomain" xlink:label="us-gaap_ClassOfWarrantOrRightDomain_default"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_SiliconValleyBankMember" xlink:label="sprb_SiliconValleyBankMember"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_DebtInstrumentExtendedTerm" xlink:label="sprb_DebtInstrumentExtendedTerm"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2022/elts/srt-2022.xsd#srt_MinimumMember" xlink:label="srt_MinimumMember"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_SecondAmendmentToLoanAndSecurityAgreementMember" xlink:label="sprb_SecondAmendmentToLoanAndSecurityAgreementMember"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_FirstTrancheTermLoanMember" xlink:label="sprb_FirstTrancheTermLoanMember"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_NumberOfSharesIssuableUponExerciseOfWarrants" xlink:label="sprb_NumberOfSharesIssuableUponExerciseOfWarrants"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_SecondTrancheTermLoanMember" xlink:label="sprb_SecondTrancheTermLoanMember"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1" xlink:label="us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_EstimatedFairValueOfWarrant" xlink:label="sprb_EstimatedFairValueOfWarrant"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_StockIssuedDuringPeriodSharesConversionOfConvertibleSecurities" xlink:label="us-gaap_StockIssuedDuringPeriodSharesConversionOfConvertibleSecurities"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_LongTermDebt" xlink:label="us-gaap_LongTermDebt"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_DebtTotalAvailable" xlink:label="sprb_DebtTotalAvailable"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_DebtAvailableFirstTranche" xlink:label="sprb_DebtAvailableFirstTranche"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_DebtAvailableSecondTranche" xlink:label="sprb_DebtAvailableSecondTranche"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_LineOfCreditFacilityCurrentBorrowingCapacity" xlink:label="us-gaap_LineOfCreditFacilityCurrentBorrowingCapacity"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_DebtInstrumentInterestRateStatedPercentage" xlink:label="us-gaap_DebtInstrumentInterestRateStatedPercentage"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_EndOfTermPaymentAmount" xlink:label="sprb_EndOfTermPaymentAmount"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_RepaymentsOfDebt" xlink:label="us-gaap_RepaymentsOfDebt"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_DebtInstrumentMaturityDate" xlink:label="us-gaap_DebtInstrumentMaturityDate"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_DebtInstrumentFrequencyOfPeriodicPayment" xlink:label="us-gaap_DebtInstrumentFrequencyOfPeriodicPayment"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_DebtInstrumentBasisSpreadVariableRateScenarioOne" xlink:label="sprb_DebtInstrumentBasisSpreadVariableRateScenarioOne"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_DebtInstrumentFrequencyOfPeriodicPaymentScenarioOne" xlink:label="sprb_DebtInstrumentFrequencyOfPeriodicPaymentScenarioOne"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_DebtInstrumentBasisSpreadVariableRateScenarioTwo" xlink:label="sprb_DebtInstrumentBasisSpreadVariableRateScenarioTwo"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_DebtInstrumentFrequencyOfPeriodicPaymentScenarioTwo" xlink:label="sprb_DebtInstrumentFrequencyOfPeriodicPaymentScenarioTwo"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_NumberOfMonthlyInstallments" xlink:label="sprb_NumberOfMonthlyInstallments"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_EndOfTermPaymentPercentage" xlink:label="sprb_EndOfTermPaymentPercentage"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_DebtInstrumentPrepaymentFeePercentage" xlink:label="sprb_DebtInstrumentPrepaymentFeePercentage"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_DeferredFinanceCostsGross" xlink:label="us-gaap_DeferredFinanceCostsGross"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_DebtInstrumentCarryingAmount" xlink:label="us-gaap_DebtInstrumentCarryingAmount"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_DebtPrincipalOutstanding" xlink:label="sprb_DebtPrincipalOutstanding"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_DebtInstrumentUnamortizedDebtIssuanceCostsAndDiscount" xlink:label="sprb_DebtInstrumentUnamortizedDebtIssuanceCostsAndDiscount"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_UnamortizedDebtIssuanceExpense" xlink:label="us-gaap_UnamortizedDebtIssuanceExpense"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_DebtInstrumentUnamortizedDiscount" xlink:label="us-gaap_DebtInstrumentUnamortizedDiscount"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_InterestExpenseDebt" xlink:label="us-gaap_InterestExpenseDebt"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_LongTermDebtAverageAmountOutstanding" xlink:label="us-gaap_LongTermDebtAverageAmountOutstanding"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_DebtInstrumentInterestRatePercentageScenarioOne" xlink:label="sprb_DebtInstrumentInterestRatePercentageScenarioOne"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_DebtInstrumentInterestRatePercentageScenarioTwo" xlink:label="sprb_DebtInstrumentInterestRatePercentageScenarioTwo"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_DebtInstrumentCovenantCompliance" xlink:label="us-gaap_DebtInstrumentCovenantCompliance"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_DebtInstrumentCovenantDescription" xlink:label="us-gaap_DebtInstrumentCovenantDescription"/>
    <link:definitionArc xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="us-gaap_DebtInstrumentTable" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_DebtInstrumentTable" xlink:to="us-gaap_DebtInstrumentAxis" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_VariableRateAxis" xlink:to="us-gaap_VariableRateDomain" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_VariableRateAxis" xlink:to="us-gaap_VariableRateDomain_default" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_TypeOfArrangementAxis" xlink:to="us-gaap_ArrangementsAndNonarrangementTransactionsMember" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_RangeAxis" xlink:to="srt_RangeMember" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_RangeAxis" xlink:to="srt_RangeMember_default" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RangeMember" xlink:to="srt_MaximumMember" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="us-gaap_WarrantMember" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_StatementEquityComponentsAxis" xlink:to="us-gaap_EquityComponentDomain" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:to="sprb_FirstAmendmentToLoanAndSecurityAgreementMember" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EquityComponentDomain" xlink:to="us-gaap_CommonStockMember" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_VariableRateDomain" xlink:to="us-gaap_PrimeRateMember" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_DebtInstrumentAxis" xlink:to="us-gaap_DebtInstrumentNameDomain" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_DebtInstrumentAxis" xlink:to="us-gaap_DebtInstrumentNameDomain_default" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentNameDomain" xlink:to="sprb_TermLoanMember" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_ClassOfWarrantOrRightAxis" xlink:to="us-gaap_ClassOfWarrantOrRightDomain" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_ClassOfWarrantOrRightAxis" xlink:to="us-gaap_ClassOfWarrantOrRightDomain_default" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_EntityDomain" xlink:to="sprb_SiliconValleyBankMember" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="sprb_DebtInstrumentExtendedTerm" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="1" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_DebtInstrumentTable" xlink:to="us-gaap_StatementEquityComponentsAxis" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="1" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RangeMember" xlink:to="srt_MinimumMember" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="1" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:to="sprb_SecondAmendmentToLoanAndSecurityAgreementMember" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="1" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentNameDomain" xlink:to="sprb_FirstTrancheTermLoanMember" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="1" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="sprb_NumberOfSharesIssuableUponExerciseOfWarrants" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="2" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_DebtInstrumentTable" xlink:to="dei_LegalEntityAxis" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="2" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentNameDomain" xlink:to="sprb_SecondTrancheTermLoanMember" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="2" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="3" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_DebtInstrumentTable" xlink:to="us-gaap_VariableRateAxis" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="3" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="sprb_EstimatedFairValueOfWarrant" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="4" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_DebtInstrumentTable" xlink:to="srt_RangeAxis" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="4" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="us-gaap_StockIssuedDuringPeriodSharesConversionOfConvertibleSecurities" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="5" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_DebtInstrumentTable" xlink:to="us-gaap_TypeOfArrangementAxis" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="5" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="us-gaap_LongTermDebt" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="6" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_DebtInstrumentTable" xlink:to="us-gaap_ClassOfWarrantOrRightAxis" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="6" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="sprb_DebtTotalAvailable" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="7" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="sprb_DebtAvailableFirstTranche" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="8" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="sprb_DebtAvailableSecondTranche" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="9" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="us-gaap_LineOfCreditFacilityCurrentBorrowingCapacity" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="10" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="us-gaap_DebtInstrumentInterestRateStatedPercentage" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="11" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="sprb_EndOfTermPaymentAmount" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="12" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="us-gaap_RepaymentsOfDebt" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="13" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="us-gaap_DebtInstrumentMaturityDate" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="14" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="us-gaap_DebtInstrumentFrequencyOfPeriodicPayment" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="15" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="sprb_DebtInstrumentBasisSpreadVariableRateScenarioOne" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="16" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="sprb_DebtInstrumentFrequencyOfPeriodicPaymentScenarioOne" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="17" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="sprb_DebtInstrumentBasisSpreadVariableRateScenarioTwo" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="18" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="sprb_DebtInstrumentFrequencyOfPeriodicPaymentScenarioTwo" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="19" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="sprb_NumberOfMonthlyInstallments" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="20" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="sprb_EndOfTermPaymentPercentage" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="21" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="sprb_DebtInstrumentPrepaymentFeePercentage" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="22" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="us-gaap_DeferredFinanceCostsGross" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="23" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="us-gaap_DebtInstrumentCarryingAmount" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="24" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="sprb_DebtPrincipalOutstanding" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="25" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="sprb_DebtInstrumentUnamortizedDebtIssuanceCostsAndDiscount" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="26" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="us-gaap_UnamortizedDebtIssuanceExpense" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="27" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="us-gaap_DebtInstrumentUnamortizedDiscount" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="28" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="us-gaap_InterestExpenseDebt" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="29" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="us-gaap_LongTermDebtAverageAmountOutstanding" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="30" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="sprb_DebtInstrumentInterestRatePercentageScenarioOne" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="31" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="sprb_DebtInstrumentInterestRatePercentageScenarioTwo" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="32" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="us-gaap_DebtInstrumentCovenantCompliance" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="33" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="us-gaap_DebtInstrumentCovenantDescription" priority="2" use="optional"/>
  </link:definitionLink>
  <link:definitionLink xlink:type="extended" xlink:role="http://www.sprucebiosciences.com/20230630/taxonomy/role/DisclosureCollaborationAndLicenseAgreementsAdditionalInformationDetails">
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:label="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_ScheduleOfResearchAndDevelopmentArrangementContractToPerformForOthersTable" xlink:label="us-gaap_ScheduleOfResearchAndDevelopmentArrangementContractToPerformForOthersTable"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_TypeOfArrangementAxis" xlink:label="us-gaap_TypeOfArrangementAxis"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="us-gaap_ArrangementsAndNonarrangementTransactionsMember"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2022/elts/srt-2022.xsd#srt_RepurchaseAgreementCounterpartyNameDomain" xlink:label="srt_RepurchaseAgreementCounterpartyNameDomain"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_EliLillyAndCompanyMember" xlink:label="sprb_EliLillyAndCompanyMember"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_UpfrontPayments" xlink:label="sprb_UpfrontPayments"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2022/elts/srt-2022.xsd#srt_CounterpartyNameAxis" xlink:label="srt_CounterpartyNameAxis"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_LillyLicenseAgreementMember" xlink:label="sprb_LillyLicenseAgreementMember"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2022/elts/srt-2022.xsd#srt_RepurchaseAgreementCounterpartyNameDomain" xlink:label="srt_RepurchaseAgreementCounterpartyNameDomain_default"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersByTypeAxis" xlink:label="us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersByTypeAxis"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersTypeDomain" xlink:label="us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersTypeDomain"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersTypeDomain" xlink:label="us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersTypeDomain_default"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_PotentialAdditionalPayment" xlink:label="sprb_PotentialAdditionalPayment"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_KakenLicenseAgreementMember" xlink:label="sprb_KakenLicenseAgreementMember"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersCostsIncurredGross" xlink:label="us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersCostsIncurredGross"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_AdditionalMilestonePayment" xlink:label="sprb_AdditionalMilestonePayment"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_ResearchAndDevelopmentExpense" xlink:label="us-gaap_ResearchAndDevelopmentExpense"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_Revenues" xlink:label="us-gaap_Revenues"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_DeferredRevenueCurrent" xlink:label="us-gaap_DeferredRevenueCurrent"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_DeferredRevenue" xlink:label="us-gaap_DeferredRevenue"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_PotentialAdditionalCashReceipt" xlink:label="sprb_PotentialAdditionalCashReceipt"/>
    <link:definitionArc xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="us-gaap_ScheduleOfResearchAndDevelopmentArrangementContractToPerformForOthersTable" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_TypeOfArrangementAxis" xlink:to="us-gaap_ArrangementsAndNonarrangementTransactionsMember" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RepurchaseAgreementCounterpartyNameDomain" xlink:to="sprb_EliLillyAndCompanyMember" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="sprb_UpfrontPayments" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfResearchAndDevelopmentArrangementContractToPerformForOthersTable" xlink:to="srt_CounterpartyNameAxis" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:to="sprb_LillyLicenseAgreementMember" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_CounterpartyNameAxis" xlink:to="srt_RepurchaseAgreementCounterpartyNameDomain" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_CounterpartyNameAxis" xlink:to="srt_RepurchaseAgreementCounterpartyNameDomain_default" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersByTypeAxis" xlink:to="us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersTypeDomain" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersByTypeAxis" xlink:to="us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersTypeDomain_default" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="1" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="sprb_PotentialAdditionalPayment" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="1" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfResearchAndDevelopmentArrangementContractToPerformForOthersTable" xlink:to="us-gaap_TypeOfArrangementAxis" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="1" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:to="sprb_KakenLicenseAgreementMember" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="2" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersCostsIncurredGross" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="2" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfResearchAndDevelopmentArrangementContractToPerformForOthersTable" xlink:to="us-gaap_ResearchAndDevelopmentArrangementContractToPerformForOthersByTypeAxis" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="3" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="sprb_AdditionalMilestonePayment" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="4" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="us-gaap_ResearchAndDevelopmentExpense" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="5" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="us-gaap_Revenues" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="6" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="us-gaap_DeferredRevenueCurrent" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="7" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="us-gaap_DeferredRevenue" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="8" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="sprb_PotentialAdditionalCashReceipt" priority="2" use="optional"/>
  </link:definitionLink>
  <link:definitionLink xlink:type="extended" xlink:role="http://www.sprucebiosciences.com/20230630/taxonomy/role/Role_DisclosureCapitalStructureAdditionalInformationDetails">
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_ClassOfStockLineItems" xlink:label="us-gaap_ClassOfStockLineItems"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_ScheduleOfStockByClassTable" xlink:label="us-gaap_ScheduleOfStockByClassTable"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_TypeOfArrangementAxis" xlink:label="us-gaap_TypeOfArrangementAxis"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="us-gaap_ArrangementsAndNonarrangementTransactionsMember"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_StatementEquityComponentsAxis" xlink:label="us-gaap_StatementEquityComponentsAxis"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_EquityComponentDomain" xlink:label="us-gaap_EquityComponentDomain"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_SecuritiesPurchaseAgreementMember" xlink:label="sprb_SecuritiesPurchaseAgreementMember"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_PreFundedWarrantsMember" xlink:label="sprb_PreFundedWarrantsMember"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_CommonStockSharesAuthorized" xlink:label="us-gaap_CommonStockSharesAuthorized"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_StandardWarrantsMember" xlink:label="sprb_StandardWarrantsMember"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_CommonStockParOrStatedValuePerShare" xlink:label="us-gaap_CommonStockParOrStatedValuePerShare"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_CommonStockVotingRights" xlink:label="us-gaap_CommonStockVotingRights"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_DividendsCommonStock" xlink:label="us-gaap_DividendsCommonStock"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_CommonStockSharesIssued" xlink:label="us-gaap_CommonStockSharesIssued"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_CommonStockPurchasableUnderPrefundedWarrants" xlink:label="sprb_CommonStockPurchasableUnderPrefundedWarrants"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_CommonStockPurchasableUnderWarrants" xlink:label="sprb_CommonStockPurchasableUnderWarrants"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1" xlink:label="us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_CommonStockSharesExercisedUnderPrefundedWarrants" xlink:label="sprb_CommonStockSharesExercisedUnderPrefundedWarrants"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_PreFundedWarrantsOutstanding" xlink:label="sprb_PreFundedWarrantsOutstanding"/>
    <link:definitionArc xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_ClassOfStockLineItems" xlink:to="us-gaap_ScheduleOfStockByClassTable" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_TypeOfArrangementAxis" xlink:to="us-gaap_ArrangementsAndNonarrangementTransactionsMember" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_StatementEquityComponentsAxis" xlink:to="us-gaap_EquityComponentDomain" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfStockByClassTable" xlink:to="us-gaap_StatementEquityComponentsAxis" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:to="sprb_SecuritiesPurchaseAgreementMember" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EquityComponentDomain" xlink:to="sprb_PreFundedWarrantsMember" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems" xlink:to="us-gaap_CommonStockSharesAuthorized" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="1" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfStockByClassTable" xlink:to="us-gaap_TypeOfArrangementAxis" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="1" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EquityComponentDomain" xlink:to="sprb_StandardWarrantsMember" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="1" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems" xlink:to="us-gaap_CommonStockParOrStatedValuePerShare" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="2" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems" xlink:to="us-gaap_CommonStockVotingRights" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="3" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems" xlink:to="us-gaap_DividendsCommonStock" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="4" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems" xlink:to="us-gaap_CommonStockSharesIssued" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="5" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems" xlink:to="sprb_CommonStockPurchasableUnderPrefundedWarrants" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="6" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems" xlink:to="sprb_CommonStockPurchasableUnderWarrants" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="7" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems" xlink:to="us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="8" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems" xlink:to="sprb_CommonStockSharesExercisedUnderPrefundedWarrants" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="9" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems" xlink:to="sprb_PreFundedWarrantsOutstanding" priority="2" use="optional"/>
  </link:definitionLink>
  <link:definitionLink xlink:type="extended" xlink:role="http://www.sprucebiosciences.com/20230630/taxonomy/role/Role_DisclosureCapitalStructureSummaryOfCommonStockReservedForFutureIssuanceDetails">
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_ClassOfStockLineItems" xlink:label="us-gaap_ClassOfStockLineItems"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_ScheduleOfStockByClassTable" xlink:label="us-gaap_ScheduleOfStockByClassTable"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_SaleOfStockNameOfTransactionDomain" xlink:label="us-gaap_SaleOfStockNameOfTransactionDomain"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_EmployeeStockMember" xlink:label="us-gaap_EmployeeStockMember"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_SubsidiarySaleOfStockAxis" xlink:label="us-gaap_SubsidiarySaleOfStockAxis"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_StatementEquityComponentsAxis" xlink:label="us-gaap_StatementEquityComponentsAxis"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_EquityComponentDomain" xlink:label="us-gaap_EquityComponentDomain"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_AwardTypeAxis" xlink:label="us-gaap_AwardTypeAxis"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_EmployeeStockOptionMember" xlink:label="us-gaap_EmployeeStockOptionMember"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_CommonStockWarrantsMember" xlink:label="sprb_CommonStockWarrantsMember"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_CommonStockCapitalSharesReservedForFutureIssuance" xlink:label="us-gaap_CommonStockCapitalSharesReservedForFutureIssuance"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_default"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_StockOptionsAvailableForFutureIssuanceMember" xlink:label="sprb_StockOptionsAvailableForFutureIssuanceMember"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_RestrictedAndPerformanceStockUnitsIssuedAndOutstandingMember" xlink:label="sprb_RestrictedAndPerformanceStockUnitsIssuedAndOutstandingMember"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_SharesAvailableForFutureIssuanceUnderTwoThousandAndTwentyEquityIncentivePlanMember" xlink:label="sprb_SharesAvailableForFutureIssuanceUnderTwoThousandAndTwentyEquityIncentivePlanMember"/>
    <link:definitionArc xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_ClassOfStockLineItems" xlink:to="us-gaap_ScheduleOfStockByClassTable" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:to="us-gaap_EmployeeStockMember" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_SubsidiarySaleOfStockAxis" xlink:to="us-gaap_SaleOfStockNameOfTransactionDomain" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_StatementEquityComponentsAxis" xlink:to="us-gaap_EquityComponentDomain" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfStockByClassTable" xlink:to="us-gaap_AwardTypeAxis" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="us-gaap_EmployeeStockOptionMember" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EquityComponentDomain" xlink:to="sprb_CommonStockWarrantsMember" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems" xlink:to="us-gaap_CommonStockCapitalSharesReservedForFutureIssuance" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_AwardTypeAxis" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_AwardTypeAxis" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_default" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="1" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfStockByClassTable" xlink:to="us-gaap_StatementEquityComponentsAxis" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="1" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="sprb_StockOptionsAvailableForFutureIssuanceMember" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="2" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfStockByClassTable" xlink:to="us-gaap_SubsidiarySaleOfStockAxis" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="2" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="sprb_RestrictedAndPerformanceStockUnitsIssuedAndOutstandingMember" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="3" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="sprb_SharesAvailableForFutureIssuanceUnderTwoThousandAndTwentyEquityIncentivePlanMember" priority="2" use="optional"/>
  </link:definitionLink>
  <link:definitionLink xlink:type="extended" xlink:role="http://www.sprucebiosciences.com/20230630/taxonomy/role/DisclosureStockbasedCompensationExpenseAdditionalInformationDetails">
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:label="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_TypeOfArrangementAxis" xlink:label="us-gaap_TypeOfArrangementAxis"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="us-gaap_ArrangementsAndNonarrangementTransactionsMember"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_VestingAxis" xlink:label="us-gaap_VestingAxis"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_VestingDomain" xlink:label="us-gaap_VestingDomain"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_VestingDomain" xlink:label="us-gaap_VestingDomain_default"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_ClinicalDevelopmentMilestonesMember" xlink:label="sprb_ClinicalDevelopmentMilestonesMember"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_RestrictedStockUnitsRSUMember" xlink:label="us-gaap_RestrictedStockUnitsRSUMember"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_AwardTypeAxis" xlink:label="us-gaap_AwardTypeAxis"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsGranted" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsGranted"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_ShareBasedCompensationAwardTrancheFourMember" xlink:label="sprb_ShareBasedCompensationAwardTrancheFourMember"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsNonvestedWeightedAverageGrantDateFairValue" xlink:label="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsNonvestedWeightedAverageGrantDateFairValue"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_ProbableOfAchievementMember" xlink:label="sprb_ProbableOfAchievementMember"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsOutstandingNumber" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsOutstandingNumber"/>
    <link:definitionArc xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_TypeOfArrangementAxis" xlink:to="us-gaap_ArrangementsAndNonarrangementTransactionsMember" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_VestingAxis" xlink:to="us-gaap_VestingDomain" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_VestingAxis" xlink:to="us-gaap_VestingDomain_default" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:to="sprb_ClinicalDevelopmentMilestonesMember" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="us-gaap_RestrictedStockUnitsRSUMember" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_AwardTypeAxis" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsGranted" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:to="us-gaap_TypeOfArrangementAxis" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_VestingDomain" xlink:to="sprb_ShareBasedCompensationAwardTrancheFourMember" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="1" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsNonvestedWeightedAverageGrantDateFairValue" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="1" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:to="us-gaap_AwardTypeAxis" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="1" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_VestingDomain" xlink:to="sprb_ProbableOfAchievementMember" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="2" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsOutstandingNumber" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="2" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:to="us-gaap_VestingAxis" priority="2" use="optional"/>
  </link:definitionLink>
  <link:definitionLink xlink:type="extended" xlink:role="http://www.sprucebiosciences.com/20230630/taxonomy/role/DisclosureStockbasedCompensationExpenseSummaryOfStockbasedCompensationExpenseDetails">
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_EmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsLineItems" xlink:label="us-gaap_EmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsLineItems"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable" xlink:label="us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_IncomeStatementLocationAxis" xlink:label="us-gaap_IncomeStatementLocationAxis"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_IncomeStatementLocationDomain" xlink:label="us-gaap_IncomeStatementLocationDomain"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_IncomeStatementLocationDomain" xlink:label="us-gaap_IncomeStatementLocationDomain_default"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_AllocatedShareBasedCompensationExpense" xlink:label="us-gaap_AllocatedShareBasedCompensationExpense"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_ResearchAndDevelopmentExpenseMember" xlink:label="us-gaap_ResearchAndDevelopmentExpenseMember"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_GeneralAndAdministrativeExpenseMember" xlink:label="us-gaap_GeneralAndAdministrativeExpenseMember"/>
    <link:definitionArc xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_EmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsLineItems" xlink:to="us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable" xlink:to="us-gaap_IncomeStatementLocationAxis" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_IncomeStatementLocationAxis" xlink:to="us-gaap_IncomeStatementLocationDomain" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_IncomeStatementLocationAxis" xlink:to="us-gaap_IncomeStatementLocationDomain_default" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsLineItems" xlink:to="us-gaap_AllocatedShareBasedCompensationExpense" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncomeStatementLocationDomain" xlink:to="us-gaap_ResearchAndDevelopmentExpenseMember" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="1" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncomeStatementLocationDomain" xlink:to="us-gaap_GeneralAndAdministrativeExpenseMember" priority="2" use="optional"/>
  </link:definitionLink>
  <link:definitionLink xlink:type="extended" xlink:role="http://www.sprucebiosciences.com/20230630/taxonomy/role/Role_DisclosureNetLossPerShareScheduleOfPotentiallyDilutiveSecuritiesNotIncludedInCalculationOfDilutedPerShareDetails">
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems" xlink:label="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable" xlink:label="us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis" xlink:label="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_AntidilutiveSecuritiesNameDomain" xlink:label="us-gaap_AntidilutiveSecuritiesNameDomain"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_AntidilutiveSecuritiesNameDomain" xlink:label="us-gaap_AntidilutiveSecuritiesNameDomain_default"/>
    <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2022/elts/us-gaap-2022.xsd#us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareAmount" xlink:label="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareAmount"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_SharesSubjectToOutstandingCommonStockOptionsMember" xlink:label="sprb_SharesSubjectToOutstandingCommonStockOptionsMember"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_SharesSubjectToOutstandingRestrictedStockUnitsMember" xlink:label="sprb_SharesSubjectToOutstandingRestrictedStockUnitsMember"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_EstimatedSharesIssuableUnderEmployeeStockPurchasePlanMember" xlink:label="sprb_EstimatedSharesIssuableUnderEmployeeStockPurchasePlanMember"/>
    <link:loc xlink:type="locator" xlink:href="sprb-20230630.xsd#sprb_SharesSubjectToOutstandingStandardWarrantsMember" xlink:label="sprb_SharesSubjectToOutstandingStandardWarrantsMember"/>
    <link:definitionArc xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems" xlink:to="us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis" xlink:to="us-gaap_AntidilutiveSecuritiesNameDomain" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis" xlink:to="us-gaap_AntidilutiveSecuritiesNameDomain_default" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems" xlink:to="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareAmount" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AntidilutiveSecuritiesNameDomain" xlink:to="sprb_SharesSubjectToOutstandingCommonStockOptionsMember" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="0" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable" xlink:to="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="1" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AntidilutiveSecuritiesNameDomain" xlink:to="sprb_SharesSubjectToOutstandingRestrictedStockUnitsMember" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="2" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AntidilutiveSecuritiesNameDomain" xlink:to="sprb_EstimatedSharesIssuableUnderEmployeeStockPurchasePlanMember" priority="2" use="optional"/>
    <link:definitionArc xlink:type="arc" order="3" xbrldt:closed="false" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AntidilutiveSecuritiesNameDomain" xlink:to="sprb_SharesSubjectToOutstandingStandardWarrantsMember" priority="2" use="optional"/>
  </link:definitionLink>
</link:linkbase>
