A number of screening tests will detect β-lactam antibiotics at levels of less than 10 ng/mL in milk. However, confirmatory procedures of comparable sensitivity are not available. A method using an automated liquid chromatography (LC) cleanup sensitive to 1 ng/mL was developed for penicillin V and cloxacillin. Milk was deproteinlzed with 2 volumes of acetonitrlle. Methylene chloride and hexane were added to the filtrate to separate the water layer containing the antibiotics. The filtrate could also be evaporated directly. The water layer was concentrated and loaded onto a polymeric LC column in 0.01 M pH 7 buffer with the aid of an autosampler. The penicillins were eluted with an acetonitrlle gradient from pH 7 buffer (100%, 0-3 mln) to pH 7 buffer-acetonltrlle (40 + 60,25 mln). Fractions containing each compound were collected and rechromatographed Isocratlcally on the same column type in 0.01 M H3P04-acetonltrlle (58 + 42 for cloxacillin; 62 + 38 for penicillin V). The cleanup can be fully automated. The approach is applicable to other penicillins, but suitable conditions for analysis of fractions for each compound must be developed. Recoveries for penicillin V were 88 ±2% at 1 ppm, 89 ±10% at 0.1 ppm, and 87 ±13% at 0.01 ppm. For cloxacillin, recoveries were 97 ±5% at 1 ppm, 90 ±4% at 0.1 ppm, and 89 ±8% at 0.01 ppm.