Two rapid (1-h) assays for the detection of Staphylococcus aureus staphylocoagulase were developed by using the fluorogenic thrombin substrates N-t-boc-Val-Pro-Arg-7-amido-4-methylcoumarin (VPA) and N-t-boc--benzyl-Asp-Pro-Arg-7-amido-4-methylocoumarin (BB). The assays were compared to the tube coagulase test and latex agglutination (LA) (Sanofi Diagnostics Pasteur, Guildford, Surrey, United Kingdom) by using 406 clinical isolates of staphylococci, and they produced positive and negative predictive values of 99.2 and 99.1% for LA, 98.9 and 92.7% for VPA, and 98.9 and 99.1% for BB. Fluorescent assays used colonies from solid media, thereby eliminating the need for broth cultures, and were performed in microtiter trays, thus making them suitable for large-scale screening.