A lytic bacteriophage 812 ranks among promising candidates for phage therapy, which emerges as an alternative to antibiotics for treatment of staphylococcal infections. Proteome of bacteriophage 812 was analyzed using various mass spectrometry techniques. Eleven proteins were identified, of which seven for the first time. Gel electrophoresis with peptide mass fingerprinting was found the most efficient for overall analysis; HPLC-ESI MS was used for confirmation in dubious cases. Seemingly abnormal migration of main tail sheath protein (812_mtsp) was explained after adjustment of gel electrophoresis conditions and peptide mass fingerprinting combined with MALDI TOF/TOF MS results. Three similarly-sized 812_mtsp fragments, which originally migrated in one band, were separated and identified. Proteome characterization of bacteriophage 812 is a prerequisite for future comparison of the standard-type phage 812 with its mutants and related phages to correlate proteome changes with their different host range.