A confirmatory assay using liquid chromatography-electrospray mass spectrometry (LC-EMS) is presented for the simultaneous determination of penicillins V and G along with oxacillin, cloxacillin and dicloxacillin in muscle, kidney and milk. Nafcillin is used as an internal standard. Samples are extracted using acetonitrile and cleaned up using liquid-liquid extraction into dichloromethane. The extracts are injected into a dedicated LC-EMS instrument and any penicillins present are detected using multiple single-ion monitoring. Three or four ions can be detected for each penicillin, meeting one of the recommended EU criteria for confirmatory residue analyses using low-resolution mass spectrometry. The detection limits are below the stipulated EU maximum residue limits for each of the penicillins.