Cancer　cell　spheroids　(CCS)　are　a　valuable　three-dimensional　cell　model　in　cancer　studies　because　they　could　replicate　numerous　characteristics　of　solid　tumors.　Increasing　researches　have　used　matrix-assisted　laser　desorption/ionization　mass　spectrometry　imaging　(MALDI-MSI)　to　investigate　the　spatial　distribution　of　endogenous　compounds　(e.g.,　lipids)　in　CCS.　However,　only　limited　lipid　species　can　be　detected　owing　to　a　low　ion　yield　by　using　MALDI.　Besides,　it　is　still　challenging　to　fully　characterize　the　structural　diversity　of　lipids　due　to　the　existence　of　isomeric/isobaric　species.　Here,　we　carried　out　the　initial　application　of　MALDI　coupled　with　laser-postionization　(MALDI-2)　and　trapped　ion　mobility　spectrometry　(TIMS)　imaging　in　HCT116　colon　CCS　to　address　these　challenges.　We　demonstrated　that　MALDI-2　is　capable　of　detecting　more　number　and　classes　of　lipids　in　HCT116　colon　CCS　with　higher　signal　intensities　than　MALDI.　TIMS　could　successfully　separate　numerous　isobaric/isomeric　species　of　lipids　in　CCS.　Interestingly,　we　found　that　some　isomeric/isobaric　species　have　totally　different　spatial　distributions　in　colon　CCS.　Further　MS/MS　imaging　analysis　was　employed　to　determine　the　compositions　of　fatty　acid　chains　for　isomeric　species　by　examining　disparities　in　signal　intensities　and　spatial　distributions　of　product　ions.　This　work　stresses　the　robust　ability　of　TIMS　and　MALDI-2　imaging　in　analyzing　endogenous　lipids　in　CCS,　which　could　potentially　become　powerful　tools　for　future　cancer　studies.　©　2024