Mass spectrometry (MS) has become a powerful and popular method to analyze macromolecules from biological systems towards the application of clinical chemistry. Disease markers related to infections can be identified with MS analysis in combination with electrophoresis or chromatographic separations. Further, direct analysis of whole pathogenic bacterial cells (taken directly from a colony) by MS can reveal specific biomarkers that can be used for taxonomy. A brief introduction to the two advanced ionization techniques, electrospray ionization and matrix-assisted laser desorption/ionization, for MS is provided in this review. Sample preparation, separation and MS-related techniques for staphylococcal proteins analysis are summarized. The review is concluded with some current clinical applications of mass spectrometry in the area of biomarker research, vaccine development, diagnosis and strain typing of infectious Staphylococci.