Toxin-antitoxin (TA) systems in bacteria typically consist of a small, labile antitoxin that binds to a stabile toxin. This association is crucial to the regulation of the toxin’s endoribonuclease activity. In E. coli, nutritional and environmental stresses are known to enhance the breakdown of the labile antitoxin, which leads to increased toxin-mediated mRNA breakdown. Several hypotheses have been advanced to describe the role of TA systems in prokaryotes, including protection of selected sections from genome loss, triggering of programmed cell death, or promoting metabolic stasis, where a cell’s growth is temporarily halted. In E. coli, antitoxins have been shown to be degraded by ATP-dependent proteases such as ClpP and Lon, which may require chaperones such as ClpA and ClpX to facilitate this processing. For example, Lon breaks down RelB of the relBE system, while both ClpPA and Lon degrade MazE of the mazEF system. However, considerably less is known about the post-translational regulation of antitoxins in Gram-positive organisms. Staphylococcus aureus encodes three known TA systems, mazEF, relBE1, and relBE2. However, it lacks the Lon protease, but has three other ATP-dependent proteases, ClpP, ClpQ and FtsH. Furthermore, it has three chaperones with similar sequence similarity to ClpA in E. coli, an important chaperone critical to the degradation of several antitoxins. To evaluate the role of ATP-dependent proteases on antitoxins in S. aureus, we examined the degradation of antitoxins MazE, RelB1 and RelB2 in strain SH1000 containing mutations in various proteases and chaperones. We demonstrate that similar to E. coli, these antitoxins are rapidly degraded in S. aureus while the associated toxin remains stable. We further show that only mutations in ClpC and ClpP increase the stability of these antitoxins, leading to longer half-lives. This finding provides a mechanism by which ClpPC serves as the functional unit to cleave antitoxins in S. aureus, and hence may be involved in regulating similar TA systems in other Gram-positive organisms.