Cellular heterogeneity plays a pivotal role in organismal physiology, influencing developmental processes, disease progression, and therapeutic responses. Single-cell metabolomics (SCM) emerges as a powerful tool to interrogate the metabolic diversity of individual cells, offering insights into cellular phenotypes beyond genomics, or transcriptomics. Recent advancements in microfluidics, automation, and image analysis have enabled minimally invasive single-cell isolation, while development of innovative mass spectrometry (MS)-based techniques has transformed metabolite detection with their high sensitivity, broad detection range, and molecular specificity. Despite challenges such as the non-amplifiable nature of metabolites and their dynamic concentration ranges like proteins, significant progress has been made in MS platforms, ionization methods, and data analysis strategies. This review highlights the latest innovations in SCM, including nano-electrospray ionization, laser desorption/ionization, and other MS techniques, alongside applications in diverse cell types such as cancer cells, plant cells, neurons, stem cells, and immune cells. Integrating SCM with orthogonal single-cell omics holds promise for systems-level understanding, with potential applications in translational and clinical research. Addressing current limitations in throughput, sensitivity, and data processing will be essential to fully unlock the potential of SCM in answering fundamental and applied biological questions.
