Staphylococcus aureus is a common pathogen known to cause relatively minor infections as well as severe disorders in humans. Although there is fair amount of published data concerning various aspects of its biology, epidemiology, genetics, etc., there is still a scarce amount of data presenting reliable and thorough investigations regarding high-throughput analysis of total S. aureus lipid content. Therefore, the aim of this study was to develop an analytical method that in combination with advanced chemometric tools enables comprehensive lipidomic analysis of S. aureus cells. The newly developed method uses high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS) to directly examine extracted lipids and further identify them within a newly developed novel S. aureus lipids database. High coverage of the S. aureus lipidome was obtained by simultaneous bacterial cell lysis and liquid-liquid extraction. The combination of three techniques enabled the analysis of the major membrane lipid classes of S. aureus: separation of the lipid extract in reversed-phase mode, Q-TOF-MS detection in positive ion mode, and lipid database mining. The developed lipidomic approach is also a powerful tool that allows one to assess the lipid content of S. aureus cells in a comparative manner between strains characterized by different phenotypic features as exemplified here by their various sensitivities toward antibiotics.