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Exploring the chemical content of individual cells not only reveals underlying cell‐to‐cell chemical heterogeneity but is also a key component in understanding how cells combine to form emergent properties of cellular networks and tissues. Recent technological advances in many analytical techniques including mass spectrometry (MS) have improved instrumental limits of detection and laser/ion probe dimensions, allowing the analysis of micron and submicron sized areas. In the case of MS, these improvements combined with MS's broad analyte detection capabilities have enabled the rise of single‐cell and single‐organelle chemical characterization. As the chemical coverage and throughput of single‐cell measurements increase, more advanced statistical and data analysis methods have aided in data visualization and interpretation. This review focuses on secondary ion MS and matrix‐assisted laser desorption/ionization MS approaches for single‐cell and single‐organelle characterization, which is followed by advances in mass spectral data visualization and analysis.