The SrtA isoform of the Staphylococcus aureus sortase transpeptidase is responsible for the covalent attachment of virulence- and colonization-associated proteins to the bacterial peptidoglycan. Sortase utilizes two substrates, undecaprenol-pyrophosphoryl-MurNAc(GlcNAc)-Ala-d-isoGlu-Lys(-Gly5)-d-Ala-d-Ala (branched Lipid II) and secreted proteins containing a highly conserved LPXTG sequence near their C termini. SrtA simultaneously cleaves the Thr-Gly bond of the LPXTG-containing protein and forms a new amide bond with the nucleophilic amino group of the Gly5 portion of branched Lipid II, anchoring the protein to this key intermediate that is subsequently polymerized into peptidoglycan. Here we show that reported fluorescence quenching activity assays for SrtA are subject to marked fluorescence inner filter effect quenching, resulting in prematurely hyperbolic velocity versus substrate profiles and underestimates of the true kinetic parameters kcat and Km. We therefore devised a discontinuous high-performance liquid chromatography (HPLC)-based assay to monitor the SrtA reaction employing the same substrates used in the fluorescence quenching assay: Gly5 and Abz-LPETG-Dap(Dnp)-NH2. Fluorescence or UV detection using these substrates facilitates separate analysis of both the acylation and the transpeptidation steps of the reaction. Because HPLC was performed using fast-flow analytical columns (<8min/run), high-throughput applications of this assay for analysis of SrtA substrate specificity, kinetic mechanism, and inhibition are now feasible. Kinetic analysis using the HPLC assay revealed that the kinetic parameters for SrtA with Abz-LPETG-Dap(Dnp)-NH2 are 5.5mM for Km and 0.27s-1 for kcat. The Km for Gly5 was determined to be 140microM. These values represent a 300-fold increase in Km for the LPXTG substrate and a 12,000-fold increase in kcat over literature-reported values, suggesting that SrtA is more a robust enzyme than previous analyses indicated.