The cell is the most basic structural unit and plays a vital role in the function of an organism. Studying the heterogeneity of cells, especially the qualitative and quantitative analyses of proteins and lipids at the cellular level and even at the subcellular level, is of great significance for the study of some important pathological or physiological processes. Due to the small size of a single cell, low content of analytes and large interference from the biological matrix within the single cell, analytical methods at the single cell level must be highly sensitive and selective. Mass spectrometry is a powerful technology for single-cell analysis, because it has high sensitivity, high selectivity and the ability to monitor multiple chemicals at the same time. In this review, four mass spectrometry-based methods applied to single-cell analysis are introduced and discussed in detail; these are electrospray ionization mass spectrometry (ESI-MS), laser desorption ionization mass spectrometry (LDI-MS), secondary ion mass spectrometry (SIMS) and inductively coupled plasma mass spectrometry (ICP-MS). The recent advances in single-cell analysis with these mass spectrometry-based techniques are summarized. We believe that this review can provide some help and reference for single-cell analysis by mass spectrometry.