The foundations of proteomics are to study gene products and their regulatory roles within cells. Paradoxically, the only evidence that sperm cells make new proteins is through mitochondrial protein synthesis. Yet despite this, spermatozoa are the perfect candidates for mass spectrometry and hence, proteomic analysis. These enterprising cells use a plethora of post-translational modifications in order to gain functionality following their production within the testis. By using a combination of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), and more recently liquid chromatography-mass spectrometry (LC-MS)/MS, recent advances in sperm cell biology, through the use of proteomics, is making unparalleled progress. The protein inventory lists being generated have shed light on transmembrane proteins, kinases and chaperones never previously recognized. In addition, the ability to isolate either phosphopeptides or glycopeptides and quantify the differences between cells of two different populations make proteomic analysis of spermatozoa a real chance to finally answer some age old questions.