Proteins are decorated with a diverse array of post-translational modifications (PTMs) that regulate their spatial and temporal functions. Recent mass spectrometry (MS)-based studies have identified hundreds of thousands of PTM sites in mammalian proteomes. However, the signaling cues and enzymes regulating individual sites are often not known and their functional roles remain uncharacterized. Quantification of PTM site stoichiometry can help in prioritizing sites for functional analyses and is important for constructing mechanistic models of PTM-dependent protein regulation. Here, we review the concept of PTM site stoichiometry, critically evaluate the merits and drawbacks of different MS-based methods used for quantifying PTM site stoichiometry, and discuss the usefulness and limitations of stoichiometry in informing on the biological function of modified sites.