Staphylococcus aureus is the major contagious bovine mastitis pathogen. It harbors several cell surface proteins that involve in the pathogenesis of mastitis. There is no effective vaccine against S. aureus mastitis. Strain variation and lack of common target antigen/s are implicated for absence of effective vaccine. The aim of this study was to extract surface proteins S. aureus strains from cases of bovine mastitis and evaluated their immunogenicity. Nine genetically distinct S. aureus strains were used in the extraction. We used cholic acid, hexadecane, and biotinylation for surface proteins extraction. After extraction, we evaluated bacterial cells integrity by Gram stain and electron microscopy. The extracted surface proteins were visualized on sodium dodecyl sulfate PAGE (SDS-PAGE) and evaluated for immunogenicity by Western blot. We used descriptive statistics for data analysis. Our results showed that the 3 extraction methods provided comparable number of protein bands on SDS-PAGE. The average visible bands were 19, 21, and 20 by hexadecane, biotinylation, and cholic acid, respectively. Western blot showed over 10 reacting surface proteins, of which 5 (18, 22, 30, 48 and 110 kDa) were conserved immunoreactive surface proteins across the 9 strains tested. Hexadecane and cholic acid induced minor damage to the bacterial cell wall as confirmed by Gram staining and scanning and transmission electron microscopy, implying minimal contamination of the surface proteins by cytosolic proteins. We concluded that any of the 3-methods are valid for the extraction of surface proteins. However, hexadecane or cholic acid methods are easy, cheap and have minor effects on bacterial cell wall. The 5 conserved immunoreactive surface proteins may serve as potential candidates for vaccine antigen to control S. aureus mastitis in dairy cows.