Sulfonamide antibiotics are applied in veterinary medicine for the treatment of microbial infections. For the detection of residues of sulfonamides in milk, a multi-sulfonamide flow cytometric immunoassay (FCI) was developed using the Luminex MultiAnalyte Profiling (xMAP) technology. In this automated FCI, a previously developed biotinylated multi-sulfonamide mutant antibody (M.3.4) was applied in combination with fluorescent beads, directly coated with a sulfathiazole derivative, and streptavidin-phycoerythrin (SAPE) for the detection. With this FCI, at least 11 different sulfonamides could be detected (more than 50% inhibition at the 100 ng mL(-1) level) and, after an incubation of 1h, measurements were rapid (10s per sample). For the application with raw milk, a 96-well microplate-based filtration step was included into the protocol to remove disturbing milk fat particles. Because of differences in sensitivity towards different sulfonamides, the FCI was considered and validated as a qualitative screening assay. For sulfadoxine, the most applied sulfonamide in Dutch dairy cattle, the detection capability (CCbeta) was <50 microg L(-1) and this level seems feasible for five other sulfonamides. For sulfadiazine, the CCbeta was <200 microg L(-1) and this level seems feasible for four other sulfonamides. A major advantage of the applied xMAP-technology, with its 100 different color-coded bead sets, is the possibility to develop multiplex immunoassays for the simultaneous detection of several antibiotics.