Proteomics is the study of the proteins of cells or tissues. Sperm proteomics aims at the identification of the proteins that compose the sperm cell and the study of their function. The recent developments in mass spectrometry (MS) have markedly increased the throughput for the identification and study of the sperm proteins. Catalogues of spermatozoal proteins in human and in model species are becoming available laying the groundwork for subsequent research, diagnostic applications, and the development of patient-specific treatments. A wide range of MS techniques is also rapidly becoming available for researchers. This chapter describes a methodological option to study the sperm cell using MS and provides a detailed protocol to identify the proteins extracted from a Percoll-purified human sperm population and separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) using LC-MS/MS.