Post-translational modifications (PTMs) provide a rapid mechanism that enables protein phenotypic diversity so that proteins can react to external and internal disturbances and regulate cellular activity. This Biotech Brief discusses tools and methods that are employed in detecting these sub-stoichiometric and elusive protein modifiers, including mass spectrometry, antibody, artificial intelligence, ubiquitin-clipping, and specific uncaging-assisted biotinylation-based approaches. The article also discusses challenges in detecting PTMs, such as the development of PTM antibodies with high specificity and binding affinity.