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A simple high-performance liquid chromatography method for the determination of cefovecin in small volume plasma has been developed. Following solid-phase extraction using Oasis HLB cartridges, samples were separated by reverse-phase high-performance liquid chromatography on an XBridge C-8 (3.5 mu m) 4.6 x 250 mm column and quantified using ultraviolet detection at 280 nm. The mobile phase was a mixture of 10 mM ammonium acetate (pH 3.5) and acetonitrile (89: 11), with a flow rate of 0.85 mL/min. The standard curve ranged from 0.1 to 200 mu g/mL. Intra-and Inter-assay variability for cefovecin was <10%, and the average recovery was >90%. The lower limit of quantitation was 0.1 mu g/mL. This method was successfully applied to the analysis of cefovecin samples at our institution. This is also the first fully validated method with an internal standard that does not use mass spectrometry. Copyright (C) 2014 John Wiley & Sons, Ltd.