Staphylococcus aureus is an overwhelming pathogen capable of thriving in virtually any tissue of the human body. Thus, it is commonly isolated from a plethora of diseases, ranging from superficial skin infections to deep-seeded, life-threatening infections. From all S. aureus infections, osteomyelitis is one of the most devastating with the high recurrence rates due to the difficulty of achieving full therapeutic effects with antibiotics alone. Chronic infections, like osteomyelitis, are a major clinical problem that requires improved outcomes for the affected individuals. To this end, understanding the pathogenic mechanisms underlying this disease is key to uncover new therapeutic targets that can be used alone or to enhance antibiotic therapy. In this dissertation, changes in the virulence factor repertoire of S. aureus associated with increased production of extracellular proteases in regulatory mutants were studied aiming at identifying loci contributing to virulence either directly, by interacting with the host, or indirectly, by modulating the accumulation of virulence factors through protease-mediated degradation. We demonstrated that virulence in a murine osteomyelitis model can be impacted by means of reduced accumulation of virulence factors due to their decreased production or increased degradation by S. aureus own extracellular proteases. Included among the virulence factors that differed the most in abundance in a manner that correlated with virulence in our osteomyelitis model were FnBPs, Coa, Sbi, Ald1 and an uncharacterized protein. Additionally, as a result of the major role that extracellular proteases play in modulating virulence we studied regulatory mutants that impact protease production in a direct manner aiming at prioritizing loci that warrant further studies and could be exploited as potential therapeutic targets due to their protease-mediated impact on the virulence factor repertoire. We confirmed that sarA plays a predominant role in this regard followed by rot, sarS and mgrA, all of which were found to have a more strain-dependent impact on protease production by comparison to sarA. Lastly, we identified S. aureus proteases that play key roles in different clinically relevant phenotypes, these being aureolysin as main contributor to cytotoxicity to osteoblasts and osteoclast-like cells and staphopains as the contributors defining biofilm formation.