Many　diseases　are　related　to　abnormal　metabolites　in　cells.　It　is　of　great　significance　to　analyze　the　metabolites　for　cellular　physiological,　pathological,　and　pharmacological　research.　Metabolite　analysis　at　the　single-cell　level　is　extremely　important　due　to　cell　heterogeneity.　Electrospray　ionization　mass　spectrometry　(ESI-MS)　has　become　an　indispensable　method　for　single-cell　metabolite　analysis　with　the　following　advantages:　detection　of　unlabeled　substrates,　in　situ　and　real-time　in　vivo　analyses,　broad-spectrum　analysis,　and　excellent　qualitative　ability.　In　this　review,　we　discuss　published　articles　in　the　field　of　single-cell　metabolite　analysis　by　ESI-MS　over　recent　years.　ESI-MS　methods　are　classified　and　reviewed　according　to　the　methods　of　obtaining　single-cell　samples.　The　achievements　with　respect　to　different　approaches　to　sensitivity,　throughput,　coverage,　and　system　stability　are　reviewed,　and　the　methods　with　great　breakthroughs　are　summarized　and　discussed.　Finally,　the　development　trend　of　single-cell　metabolite　analysis　by　ESI-MS　is　prospected.　(C)　2021　Elsevier　B.V.　All　rights　reserved.