Post-translational modifications (PTMs) regulate the activity and macromolecular interactions of many proteins. To date over 300 PTMs have been described, and many of them play essential roles in the pathogenesis of diseases. With recent advances in biological mass spectrometry (MS), comprehensive and accurate measurements of PTMs at the proteome scale has become possible, allowing a better understanding of cellular processes and disease mechanisms. Here we review recent proteomics methods for detection of some of the biologically important PTMs, including acetylation, glycosylation, S-nitrosylation, phosphorylation, and ubiquitylation.