Through TMT-based quantitative proteomic profiling of mouse condensing and elongating spermatids, we identified 3,901 proteins and applied GO annotation to screen for hydrolase-related candidates. Among 51 candidate hydrolases, five proteins showed nuclear localization annotations, suggesting that a subset of nuclear hydrolytic enzymes may participate in the regulation of spermiogenic chromatin remodeling. These findings provide a candidate framework for exploring enzymes that potentially mediate chromatin protein turnover, protamine maturation, and sperm nuclear condensation during late spermiogenesis.