The area of single-cell analysis presents multiple challenges, including very limited sample amounts (e.g., the volume of a typical mammalian cell ranges between 1 and 10 pL) and extremely complex compositions (e.g., ~2-4 million proteins/μm 3 and >42,000 metabolites in a cell) . Omics endeavors to thoroughly characterize all elements of cellular systems. Numerous cutting-edge technologies have been employed to study genomics , epigenomics , transcriptomics , proteomics , and metabolomics at the single-cell level. The metabolome, encompassing the entirety of a cell's metabolites, emerges as a sensitive response to cell status and alterations in its surroundings. Unlike genes and proteins, which represent the cell's potential capabilities, the metabolome has more rapid (e.g., within a few seconds) response to environmental perturbations . Studying the metabolome provides a unique lens into the immediate impact of environmental changes on the cell's functional state, offering insights that extend beyond the capabilities of genomic and proteomic analyses. Thus, in addition to above-stated challenges (i.e., extremely limited sample amount and complex compositions), metabolomics studies of single cells, particularly for cells in their living status, need to minimize the influence of rapid turnover rates on profiles of cell metabolites during data acquisition. Multiple techniques, including nuclear magnetic resonance (NMR) spectroscopy, fluorescence microscopy , and mass spectrometry (MS), are commonly used for conventional metabolomics studies. Among them, MS-based methods are more effective for single cell metabolomic analysis due to its unique advantages: highly sensitive for detection and highly accurate for identification of extremely low abundance molecules with complex compositions . Several types of single-cell MS (SCMS) methods, categorized as either vacuum-based or ambient-based techniques, according to their sampling and ionization conditions, have been created and utilized for examining various cell types, including plant cells, mammalian cells, and yeasts. Vacuum-based SCMS methods predominantly rely on two approaches: secondary ion mass spectrometry (SIMS) and matrix-assisted desorption/ionization (MALDI) mass spectrometry. These techniques employ highenergy ion beams (for SIMS) or ultraviolet (UV) laser pulses (for MALDI-MS) to desorb and ionize cellular molecules, including metabolites, lipids, and pharmaceuticals, enabling sensitive and consistent analysis at the individual cell level. Unlike vacuum-based methods, ambient SCMS techniques enable analysis of cells with minimal or no sample preparation . Various ambient SCMS methods have been developed, including laser ablation electrospray ionization (LAESI) MS , live single-cell video-MS, induced nanoESI (InESI) MS , nano-spray desorption electrospray ionization (nano-DESI) MS , probe electrospray ionization (PESI) , and methods integrated with microfluidic chips and flow cytometry . We have developed the Single-probe, a multifunctional device that can be coupled to MS for single cell studies , MS imaging of tissues , and analysis of extracellular molecules within live spheroids in ambient environment. In addition, we have created the T-probe and micropipette capillary for SCMS measurements. These methodologies offer significant potential for exploring basic cell biology (e.g., cell heterogeneity , cell-cell interactions , and influence of environment on cell metabolism ) and potential clinical applications (e.g., quantification of drug and signaling molecules in single cells , drug resistance , and drug influence on cell metabolism ). Among them, the Single-probe SCMS technique is routinely used for our SCMS metabolomics studies of live cells.