A simple and sensitive method was developed for determination of cephapirin and its metabolite, desacetylcephapirin, in milk. For extraction/deproteinization, 2 mL 0.2M tetraethylammonium chloride and 28 mL acetonitrile are added to 10 mL milk, and 20 mL clear filtrate (= 5 mL milk) is collected. Filtrate is evaporated to 1-2 mL and made up to 4 mL with water, filtered, and transferred to 4 mL autosampler vials. For cleanup, 2 mL filtrate is loaded onto a Supelcosil LC-18 column in 0.01M KH2PO4 (A) using a Waters WISP autosampler. The column is eluted with an acetonitrile (B) gradient from 100% A (0-3 min) to 70%A:30%B (24 min). Fractions corresponding to cephapirin and desacetylcephapirin are collected. A 0.02M pH 2.26 buffer of 0.01M decanesulfonate-acetonitrile was used for cephapirin (80 + 20) and 0.02M pH 1.96 buffer of 0.01M decanesulfonate-acetonitrile was used for desacetylcephapirin (84 + 16) on a Polymer Laboratories PLRP-S column. Recoveries at 0.01-10 ppm were 91-98%; estimated detection limits were near 2 ppb and were comparable to sensitive screening tests.