Recombinant bovine somatotropin (rbST) can be used to enhance milk production in dairy cattle. This is permitted in several countries but unauthorized in the European Union. Antibodies, which are produced endogenously in response to rbST administration, can be detected as a biomarker for indicating rbST (ab)use. For the first time, a fast and easy-to-perform screening assay for anti-rbST antibodies has been developed and applied to raw milk samples. This flow cytometric immunoassay (FCIA) is capable of discriminating between milk from rbST-treated animals and untreated animals. In accordance with literature, 67% of the rbST-treated animals responded positively with antibody production, whereas 94% of the untreated animals did not. The analysis of simulated tank milk samples showed more than 95% of the milk mixtures as truly positive for rbST treatment, indicating that the 33% physiologically non-responding cows will not be a problem when pooled tank milk samples are considered. FCIA biomarker responses in raw milk were specific for rbST and also obtained in pasteurized milk of rbST-treated animals. Using milk as a sample matrix for detection has the advantages of non-invasive sampling, and for tank milk analysis at the farm only one milk sample is needed to screen the whole farm for rbST (ab)use.