Owing to its multiple advantages (e.g., high sensitivity, high accuracy, and broad molecular coverage), mass spectrometry (MS) is regarded as one of the most important techniques for molecular analysis. With the recent advancement, a variety of different single-cell MS (SCMS) methods have been established as powerful tools to analyze large (e.g., proteins) and small (e.g., metabolites) within individual cells . Metabolomics is the study of metabolites, which are broadly defined small molecules with molecular weight <1,500 amu such as lipids, fatty acids, peptides, amino acids, nucleic acids, sugars and organic acids . The Single-probe, a microscale sampling and ionization device, can be coupled to a mass spectrometer for SCMS metabolomics studies of live single cells without complex sample preparation or labeling. The interest in the Single-probe SCMS stems from its potential to deepen our understanding of single-cell metabolism, driving both fundamental research and clinical applications forward. Notable innovations include integration with microscopy methods (e.g., fluorescence microscopy) and combination with chemical reactions, greatly extending the application of this technique. As a multifunctional device, the Single-probe can be coupled to mass spectrometry for studies in multiple different areas, including single-cell metabolomics , MS imaging (MSI) of tissue slices , and the analysis of extracellular molecules in live single spheroids, within ambient conditions. Additionally, we have developed other techniques, such as the T-probe and micropipette capillary , to facilitate SCMS measurements. As a robust technique, the Single-probe SCMS method has been used in a variety of fundamental studies such as analysis of cellular heterogeneity , investigation of cell-cell interactions , detection of signaling molecules and environmental influences on cell metabolism. In addition, the Single-probe SCMS technique has promising clinical applications, as it has been implemented to detecting and quantifying drug molecules in single cells, characterizing cancer stem cells, investigating drug resistance and metabolic responses to drugs , and studying human diseases . In addition to mammalian cells, the Single-probe SCMS technique has been used to study plant cells This review aims to explore recent advancements and applications of the Single-probe SCMS techniques, underscoring its growing significance in analytical chemistry. As this technology continues to evolve, it promises to usher in a new era in microscale bioanalysis, enabling unprecedented insights into complex biological systems at the cellular and tissue levels.