In the important human pathogen Staphylococcus aureus the cytoplasmic ClpP protease is essential for mounting cellular stress responses and for virulence. To directly identify substrates of the ClpP protease, we expressed in vivo a proteolytic inactive form of ClpP (ClpP(trap)) that will retain but not degrade substrates translocated into its proteolytic chamber. Substrates captured inside the proteolytic barrel were co-purified along with the His-tagged ClpP complex and identified by mass spectrometry. In total, approximately 70 proteins were trapped in both of the two S. aureus strains NCTC8325-4 and Newman. About one-third of the trapped proteins are previously shown to be unstable or to be substrates of ClpP in other bacteria, supporting the validity of the ClpP-TRAP. This group of proteins encompassed the transcriptional regulators CtsR and Spx, the ClpC adaptor proteins McsB and MecA, and the cell division protein FtsZ. Newly identified ClpP substrates include the global transcriptional regulators PerR and HrcA, proteins involved in DNA damage repair (RecA, UvrA, UvrB), and proteins essential for protein synthesis (RpoB and Tuf). Our study hence underscores the central role of Clp-proteolysis in a number of pathways that contribute to the success of S. aureus as a human pathogen.