Streptococcus pyogenes (group A Streptococcus, GAS) is a pathogen that causes necrotizing fasciitis and toxic shock syndrome. Necrotizing fasciitis, a disease characterized by severe tissue destruction, and toxic shock syndrome, a systemic response to infection, result in significant mortality. GAS isolates with spontaneous inactivating mutations in the CovR/CovS two-component regulatory system are frequently isolated from patients who present with these conditions; therefore, CovR/CovS-inactivated mutations are positively correlated with invasive GAS infection. CovR/CovS-inactivated mutants frequently exhibit an encapsulated/mucoid phenotype as a result of the derepression of capsule synthesis. However, in this study, we found that 69% (20/29) of CovR/CovS-inactivated mutants did not demonstrate a distinguishable encapsulated colony morphology. In lieu of encapsulation as a marker, we identified and verified that the endopeptidase PepO can be potentially used to distinguish between CovR/CovS-activated and -inactivated isolates via sodium dodecyl-sulfate polyacrylamide gel electrophoresis and western blot hybridization. RocA is the upstream regulator of CovR/CovS, and we demonstrated that PepO expression was upregulated in the RocA-inactivated mutant isolates, including emm3-type GAS isolates (with truncated RocA). This study revealed that PepO could be a candidate marker of invasive GAS isolates with defects in the CovR/CovS pathway, informing the development of rapid detection methods to identify invasive GAS isolates.