The Gram positive bacterium Staphylococcus aureus plays an important role as an opportunistic pathogen and causative agent of nosocomial infections. As research gained insight into host specific adaptation and a broad range of virulence mechanisms, S. aureus evolved as a model organism for human pathogens. Hence, the investigation of staphylococcal proteome expression and regulation supports the understanding of the pathogenicity and relevant physiology of this organism. This study focused on the analysis of protein regulation by reversible protein phosphorylation, in particular on arginine residues. Therefore, both proteome and phosphoproteome of S. aureus COL wild type were compared with the arginine phosphatase deletion mutant S. aureus COL ΔptpB under control and stress conditions in a quantitative manner. A gel-free approach, adapted to the special challenges of arginine phosphorylation, was applied to analyze the phosphoproteome of exponential growing cells after oxidative stress caused by sublethal concentrations of H2O2. Together with phenotypic characterization of S. aureus COL ΔptpB, this study disclosed first insights into the physiological role of arginine phosphorylations in Gram positive pathogens. The spectral library based quantification of phosphopeptides finally allowed to link arginine phosphorylation to staphylococcal oxidative stress response, amino acid metabolism and virulence.