In single‐molecule mass spectrometry, the mass of each ion is measured individually; making it suitable for the analysis of very large, heterogeneous objects that cannot be analyzed by conventional means. A range of single‐molecule mass spectrometry techniques has been developed, including time‐of‐flight with cryogenic detectors, a quadrupole ion trap with optical detection, single‐molecule Fourier transform ion cyclotron resonance, charge detection mass spectrometry, quadrupole ion traps coupled to charge detector plates, and nanomechanical oscillators. In addition to providing information on mass and heterogeneity, these techniques have been used to study impact craters from cosmic dust, monitor the assembly of viruses, elucidate the fluorescence dynamics of quantum dots, and much more. This review focuses on the merits of each of these technologies, their limitations, and their applications. © 2016 Wiley Periodicals, Inc. Mass Spec Rev 36:715–733, 2017