Technical Abstract: Staphylococcal enterotoxins (SEs) are a leading cause of food poisoning. They function both as toxins that cause gastroenteritis after ingestion and as superantigens that non-specifically activate large numbers of T cells. Monkey or kitten bioassays were historically developed for analysis of SE activity. To overcome the inherent limitations of such bioassays, this study describes an alternative in vitro method, a splenocyte proliferation assay that measures the superantigen activity of Staphylococcal enterotoxin A (SEA). After incubation of splenocytes with SEA, cell proliferation was measured by labeling the DNA with 5-bromo-2-deoxyuridine (BrdU) and determining BrdU incorporation by spectroscopic measurement. BrdU incorporation is shown to be highly correlated with SEA concentration (R^2=0.99) and can be used to detect 20 pg/ml of SEA, a limit of detection far lower than reported for most ELISAs. Our assay can also distinguish between active toxin and inactive forms of the toxin in foods (milk, chicken and beef). The use of immunomagnetic beads to capture and concentrate the toxin overcame food matrix interference. These results suggest that our in vitro cell-based assay is an advantageous, practical alternative to bioassay for measuring the activity of SEA and possibly other SEs in foods.