The problem of the male reproductive system has both medical and social importance. It is highlighted that spermatozoa or seminal plasma are suitable samples for proteomic analysis of the male reproductive system. We suggested that the ejaculate could serve as one of the analytic specimens to determine fertility, which obtainable with relative ease and which contains many proteins. Moreover, the testis-specific proteins could form the basis of protein panel for detecting disorders associated with male infertility. The current study aimed to examine the ejaculate proteome compared to the spermatozoa and seminal plasma proteomes to determine a panel of proteins that can be used to assess sperm quality. Our results showed that the ejaculate represented a productive source for understanding the male reproductive physiology and could serve as a potential origin for the identification of novel protein biomarkers related to spermatozoa function in infertile. We believe that proteomic analysis based on testis-specific proteins of ejaculate along with spermatozoa (or even instead) can be useful for distinguishing the fertile, sub fertile, and infertile men.