Detection of staphylococcal toxins presents a great interest for medical diagnostics. Screening of clinical samples for the presence of several types of staphylococcal toxins using traditional methods-biological tests on animals or cell cultures as well as ELISA-is laborious. Multiplex detection methods would simplify testing. We have designed an xMAP-based assay to detect three staphylococcal toxins-enterotoxins A and B (SEA and SEB) and toxic shock syndrome toxin (TSST)-in cultural supernatants obtained from different strains of Staphylococcus aureus. The limits of detection of SEA, SEB, and TSST multiplex detection in S. aureus growth medium were 10, 1,000, and 5 pg/mL, respectively. Fifty-nine samples of S. aureus cultural supernatants were tested with the xMAP assay. The developed assay has proved highly effective detection of the natural toxins in the samples obtained due to bacterial cells cultivation. In prospect, the developed test system can be used in clinical diagnostics and in monitoring of foodstuffs and environmental objects.