Methods for the measurement of penicillin concentration in bovine plasma, kidney and urine were developed and validated. Detection was based on liquid chromatography/tandem mass spectrometry (LC/MS/MS). Phenethecillin was used as an internal standard. Plasma was extracted with acetonitrile using a method with a calculated limit of quantitation (LOQ) of 12ng/mL. Kidney samples were homogenized in water and acetonitrile, then cleaned up on C18-bonded silica SPE cartridges. The LOQ of this procedure was 10ng/g. Urine samples were diluted, filtered, and analyzed directly. The LOQ of this procedure was 63ng/mL. The overall accuracy for plasma was 103% with coefficient of variation (CV) of 3%; for kidney, 96% and 11%, respectively, and for urine, 98% and 4%, respectively. These methods were applied to the analysis of plasma, urine, and kidney biopsy samples taken from standing animals that had been dosed with penicillin.