Staphylococcus aureus is a major cause of infections worldwide, and infection results in a variety of diseases. As of no surprise, protein phosphorylation is an important game player in signaling cascades and has been shown to be involved in S. aureus virulence. Albeit long neglected, eukaryotic-type serine/threonine kinases in S. aureus have been implicated in this complex signaling cascades. Due to the substoichiometric nature of protein phosphorylation and a lack of suitable analysis tools, the knowledge of these cascades is, however, to date, still limited. Here, were apply an optimized protocol for efficient phosphopeptide enrichment via Fe-IMAC followed by LC-MS/MS to get a better understanding of the impact of protein phosphorylation on the complex signaling networks involved in pathogenicity. By profiling a serine/threonine kinase and phosphatase mutant from a methicillin-resistant S. aureus mutant library, we generated the most comprehensive phosphoproteome data set of S. aureus to date, aiding a better understanding of signaling in bacteria. With the identification of 3800 class I p-sites, we were able to increase the number of identifications by more than 21 times compared with recent literature. In addition, we were able to identify 74 downstream targets of the only reported eukaryotic-type Ser/Thr kinase of the S. aureus strain USA300, Stk1. This work allowed an extensive analysis of the bacterial phosphoproteome and indicates that Ser/Thr kinase signaling is far more abundant than previously anticipated in S. aureus.