The aim of this study was to evaluate a 3-min assay Betastar® Combo 3.0) for the detection of Penicillin G, Amoxicillin, Cefazolin and Oxytetracycline hydrochloride in individual ewe milk. Antibiotics-free samples were obtained throughout three trials from three Sarda ewes. At the laboratory, samples were spiked with the four antimicrobial molecules at different concentrations. These were on the basis of the specific maximum residue limits fixed by the European Union (MRLs): 4 g/kg for Penicillin G, 4 g/kg for Amoxicillin, 50 g/kg for Cefazolin and 100 g/kg for Oxytetracycline. Negative control samples (180 tests) were always detected as negative. The limit of detection for Penicillin was lower than the MRL, whereas for Amoxicillin and Oxytetracycline it was slightly higher than the MRL in one case out of nine examinations. The limit of detection for Cefazolin was higher than the MRL. In conclusion, Betastar® Combo 3.0 was suitable for rapid screening of individual sheep milk spiked with Penicillin G, Amoxicillin and Oxytetracycline.