A liquid chromatographic procedure is described for determination of ceftiofur (CEF) residues in milk. Milk samples were diluted with ammonium acetate solution and extracted on a C18 solid-phase extraction (SPE) column. After the analyte was eluted from the SPE column with methanol, extract volumes were reduced under nitrogen, diluted to 2.0 mL with acetate buffer, and filtered. CEF was determined after separation of milk components by reverse-phase chromatography with UV detection at 293 nm. Recoveries of CEF from bovine milk fortified at 25, 50, and 100 ppb were 86.1, 90.8, and 92.0%, respectively, with coefficients of variation (CVs) of 6.4, 7.3, and 3.9%, respectively. Values of CEF obtained from analysis of milk containing 2 levels of biologically incurred residues were 26.1 and 67.3 ppb with CVs of 3.8 and 4.4%, respectively. The limits of detection and quantitation were estimated to be 4 and 7 ppb, respectively.