The aim of this study was to develop a rapid and sensitive method for the quantification of cefquinome in animal plasma and bronchoalveolar lavage (BAL) fluid using high-performance liquid chromatography combined with electrospray tandem mass spectrometry (LC-ESI-MS/MS). Cefadroxil is used as internal standard. For plasma, the sample preparation includes a simple deproteinization step with a Microcon® filter. This allows detecting the unbound cefquinome concentration, which is correlated with the concentration in other body fluids, such as BAL fluid. To be able to detect the total plasma concentration, deproteinization with acetonitrile, followed by a back-extraction of actonitrile with dichloromethane was performed. The BAL fluid is centrifuged to precipitate floating particles. Chromatographic separation is achieved on a PLRP-S column using 0.005% formic acid and methanol as mobile phase. For plasma, good linearity was observed in the range of 5–2500 ng ml−1 for both the unbound and total concentration. The response in BAL fluid was linear in the range of 4–1000 ng ml−1. The limit of quantification (LOQ) was set at 5.00 ng ml−1 for plasma and at 4.00 ng ml−1 for BAL fluid. The limit of detection (LOD) was 3.12 ng ml−1 and 0.41 ng ml−1 for the unbound and total concentration in plasma, respectively, and was 1.43 ng ml−1 for BAL fluid. The method was shown to be of use in a pharmacokinetic study in pigs, where the correlation between cefquinome concentrations in plasma and BAL fluid of pigs was studied. Copyright © 2007 John Wiley & Sons, Ltd.