Matrix-assisted　laser　desorption/ionization　time-of-flight　mass　spectrometry　imaging　(MALDI-TOF　MSI)　is　rapidly　maturing　as　an　innovative　technique　for　spatial　molecule　(m/z　＞　1000　Da)　profiling.　However,　direct　identification　of　low-molecular-weight　compounds　(m/z　＜　600　Da)　by　MALDI-TOF　MSI　using　conventional　organic　matrixes　remains　a　challenge　because　of　ionization　suppression　and　serious　matrix-related　background　interference.　Furthermore,　the　heterogeneous　cocrystallization　that　is　inherent　to　organic　matrixes　can　degrade　spatial　resolution　in　MSI.　Herein,　we　developed　a　negative　ion　surface-assisted　laser　desorption/ionization　time-of-flight　mass　spectrometry　imaging　(SALDI-TOF　MSI)　protocol　to　detect　bisphenol　S　(BPS)　and　map　its　spatial　distribution　in　mouse　tissues　by　applying　nitrogen-　and　sulfur-co-doped　carbon　dots　(N,S-co-doped　CDs)　as　a　new　matrix　through　spraying.　The　SALDI-TOF　MS　and　imaging　parameters,　such　as　matrix　concentration,　ionization　mode,　and　matrix　deposition,　were　optimized　to　improve　imaging　performance.　In　comparison　to　organic　matrixes,　the　use　of　N,S-co-doped　CDs　in　negative　ion　mode　exhibited　free　matrix　background　interference,　enhanced　MS　signal　intensity,　and　provided　high　spatial　resolution　(acquired　at　∼50　μm)　in　the　analysis　of　BPS,　which　allowed　sensitive　detection　of　the　target　compound　on　the　surfaces　of　tissue　sections.　Quantitative　assessment　was　also　made　by　spotting　BPS　standards　directly　onto　the　tissue　surface,　and　a　good　correlation　between　the　color　change　and　BPS　concentrations　was　found.　The　corresponding　detection　limit　as　low　as　the　∼pmol　level　for　BPS　was　observed　with　the　direct　visualization　from　MS　images.　Furthermore,　the　feasibility　of　the　proposed　SALDI-TOF　MSI　method　was　extended　for　in　situ　identification　of　exogenous　BPS　in　the　different　tissues　of　mouse　involving　liver,　kidney,　spleen,　and　heart　for　exposure　and　profiling　its　spatial　localization　at　different　administration　times.　In　addition,　the　general　applicability　of　the　proposed　method　was　also　evaluated　by　SALDI-TOF　MSI　analysis　of　BPAF　in　tissues.　These　successful　applications　of　SALDI-TOF　MSI　not　only　demonstrated　its　promising　potential　as　an　alternative　to　MALDI-TOF　MSI　in　profiling　small　molecules　in　tissue　sections　but　also　provided　tremendous　insight　into　the　assessment　of　BPS　exposure.　©　Copyright　2018　American　Chemical　Society.