The majority of Staphylococcus aureus virulence- and colonization-associated surface proteins contain a pentapeptide recognition motif (LPXTG). This motif can be recognized and cleaved by sortase A (SrtA) which is a membrane-bound transpeptidase. After cleavage these proteins are covalently incorporated into the peptidoglycan. Therefore, SrtA plays a key role in S. aureus virulence. We aimed to generate a substrate mimicking this SrtA recognition motif for several purposes: to incorporate this substrate into the S. aureus cell-wall in a SrtA-dependent manner, to characterize this incorporation and to determine the effect of substrate incorporation on the incorporation of native SrtA-dependent cell-surface-associated proteins. We synthesized substrate containing the specific LPXTG motif, LPETG. As a negative control we used a scrambled version of this substrate, EGTLP and a S. aureus srtA knockout strain. Both substrates contained a fluorescence label for detection by FACScan and fluorescence microscope. A spreading assay and a competitive Luminex assay were used to determine the effect of substrate treatment on native LPXTG containing proteins deposition in the bacterial cell-wall. We demonstrate a SrtA-dependent covalent incorporation of the LPETG-containing substrate in wild type S. aureus strains and several other Gram-positive bacterial species. LPETG-containing substrate incorporation in S. aureus was growth phase-dependent and peaked at the stationary phase. This incorporation negatively correlated with srtA mRNA expression. Exogenous addition of the artificial substrate did not result in a decreased expression of native SrtA substrates (e.g. clumping factor A/B and protein A) nor induced a srtA knockout phenotype.