A coordinated three-phase method was developed to determine residues of penicillin G, ampicillin, cephapirin and cloxacillin, the four β-lactam antibiotics most frequently used in mastitis preparations, in milk. An agar well diffusion technique in bioassay trays with Bacillus stearothermophilus as the test organism was used for preliminary screening. Positive samples were subjected to thin-layer chromatography followed by bioautography, and the residues were identified. An agar well diffusion method with standard levels of the specific β-lactam antibiotics was used for quantitation.