Staphylococcus aureus causes different pathologies in humans and animals. In particular, it is involved in intramammary infections in cows, causing economic losses and milk-safety problems. Although it is well-known that surface components (proteins and capsular polysaccharides) and exotoxins are virulence factors involved in the pathogenesis of bovine mastitis, less is known about the precise biochemical identity of such molecules. Therefore, mapping of surface proteins using specific disease- and environment-isolates provides a benchmark for strain comparison of pathogens with different pathogenic characteristics and antibiotic resistance mechanism and can aid in defining specific vaccine and therapeutic targets. In this study, we used a proteomic approach on protein extracts of lysostaphin-treated S. aureus in isotonic conditions, to produce a reproducible and well resolved 2-D electrophoresis (2-DE) reference map of surface associated proteins of isolated S. aureus from a case of bovine mastitis. The most abundant protein components were identified by Matrix assisted laser desorption ionisation-time of flight (MALDI-TOF) mass spectrometry.