Lipids　and　metabolites　are　small　biological　molecules　that　act　major　roles　in　cellular　functions.　Multicellular　tumor　spheroids　(MCTS)　are　a　highly　beneficial　three-dimensional　cellular　model　for　cancer　research　due　to　their　ability　to　imitate　numerous　characteristics　of　tumor　tissues.　Increasing　studies　have　performed　spatial　lipidomics　and　metabolomics　in　MCTS　using　matrix-assisted　laser　desorption/ionization　mass　spectrometry　imaging　(MALDI-MSI).　However,　these　approaches　often　lack　the　sensitivity　and　specificity　to　offer　a　comprehensive　characterization　of　lipids　and　metabolites　within　MCTS.　In　this　study,　we　addressed　this　challenge　by　utilizing　MALDI　combined　with　laser-induced　postionization　(MALDI-2)　and　trapped　ion　mobility　spectrometry　(TIMS)　imaging　in　H295R　adrenocortical　MCTS.　Our　results　showed　that　MALDI-2　could　detect　more　lipids　and　metabolites　in　MCTS　than　the　traditional　MALDI.　TIMS　data　revealed　a　successful　separation　of　many　isomeric　and　isobaric　ions　of　lipids　and　metabolites　with　different　locations　(e.g.,　proliferative　region　and　necrotic　region)　within　MCTS,　suggesting　an　enhanced　peak　capacity　for　spatial　lipidomics　and　metabolomics.　To　further　identify　these　isomeric　and　isobaric　ions,　we　performed　MS/MS　imaging　experiments　to　compare　the　differences　in　signal　intensities　and　spatial　distributions　of　product　ions.　Our　data　highlight　the　strong　potential　of　MALDI-2　and　TIMS　imaging　for　analyzing　lipids　and　metabolites　in　MCTS,　which　may　serve　as　valuable　tools　for　numerous　fields　of　biological　and　medical　research.　©　2023　Elsevier　B.V.