The Single-probe is a sophisticated analytical tool composed of several integral components that work together to facilitate microscale bioanalysis. Here, we provide a review of its fabrication as well as applications in SCMS analysis of small molecules (i.e., semi-quantitative analysis, quantitative analysis, integration with chemical reactions, evaluation of single cell sample preparation, and combined advanced data analysis), MSI of biological tissues, MS analysis of extracellular molecules in live spheroids, and other studies performed using the Single-probebased techniques.