The importance of post-translational modifications (PTMs) of proteins has become evident in the proteomic era as it plays a critical role in modulating cellular function, and can vary in response to different stimuli thereby tuning cellular mechanisms. Assessment of PTMs on a proteomic scale is a challenging task since they are substoichiometric, transient and reversible. Moreover, the amount of post-translationally modified proteins is generally very small when compared to their unmodified counterparts. Existing methodologies for identification of PTMs essentially relies on enrichment procedure to selectively increase the amount of modified peptides. These procedures need to be integrated with sophisticated mass spectrometric methods to enable the identifications of PTMs. Although the strategies developed so far are not optimal, a number of examples will be given where the combination of innovative separation methods along with advanced mass spectrometric analyses provide positive results. These experiences are leading the way for the next generation of proteomic approaches for identification of a wide range of PTMs.