Abstract Testis, the only organ responsible for generating sperm, is by far the organ with the largest variety of proteins and tissue-specific proteins in humans. In testis, spermatogenesis is a multi-step complex process well-accepted that protein and mRNA are decoupled in certain stages of spermatogenesis. With the fast development of mass spectrometry-based proteomics, it is possible to systemically study protein abundances and modifications in testis and sperm to help us understand the molecular mechanisms of spermatogenesis. This review provides an overview of the recent progress of proteomics analysis on spermatogenesis, including protein expression and multiple post-translational modifications, such as phosphorylation, glycosylation, ubiquitylation, and acetylation.