This contribution presents an overview of the potential capabilities of evolving methodologies to characterize seminal protein profiles and identify interactions while simultaneously analyzing the complex but comprehensive protein matrix. The traditional approach to protein characterization in fish semen is based on two major steps: the purification of proteins using numerous liquid preparative chromatographic methods and the identification of proteins using sequencing. In using this approach major proteins of fish seminal plasma were identified, including apolipoprotein A-I, transferrin, α1 – antiproteinase and parvalbumins for carp; and fetuin B-like protein, α1 – antiproteinase, lipocalin-type protein and apolipoprotein C-I for rainbow trout. Proteomics provides for a great advance in protein studies. Proteomics is concerned with the study of protein products expressed by the genome, with the term ‘proteome’ originating from PROTEin complement of the genOME. Proteomics emerged as a leading technology in the post-genomic era because of the central role of proteins and protein–protein interactions in cell physiology. The first applications of proteomic research in the study of fish reproduction have already been made, and include the effects of cryopreservation on semen proteome, molecular mechanisms determining sperm motility initiation and the effects of domestication of cultured species on semen quality. The introduction of proteomics in the study of male reproduction in fish creates a unique opportunity to unravel, using an integrative approach, the physiological mechanisms important for sperm function, such as motility and fertilizing ability. It is anticipated that proteomic studies will greatly increase the chances for reliably identifying new biomarkers in male fertility studies. However, a better understanding on the opportunities and limitations of the new disciplines or subject areas are required.