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A liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method was developed, using testosterone-d(3) as a surrogate analyte, for the simultaneous quantification of goserelin and testosterone in rat plasma. According to this method, the pharmacokinetic and pharmacodynamic data were obtained from a single plasma sample aliquot. The method involved the addition of alarelin as an internal standard (IS) for goserelin and testosterone-C-13(3) for testosterone or testosterone-d(3). The conditions for the separation of these two compounds were achieved on a ZORBAX Eclipse Plus C-18 column (Agilent, 2.1 x 50 mm, 1.8 mu m, Stockport, UK) in a single chromatographic run at a flow rate of 400 mu L/min. In order to minimize interferences of complex matrix, the extraction of plasma consisted of a protein precipitation step using methanol, followed by purification using an Oasis (R) HLB solid-phase extraction column. The method was validated in the concentration range of 0.01-30.0 ng/mL for goserelin and 0.05-30.0 ng/mL for testosterone-d(3), respectively. The within- and between-run precisions were 1.7-9.2% and 2.1-6.9%, respectively. The within- and between-run accuracies were -1.8 to 5.3% and -4.9 to 4.0%, respectively. This accurate and highly specific assay provides a useful method to evaluate the pharmacokinetics and pharmacodynamics of goserelin in rats. (C) 2014 Elsevier B.V. All rights reserved.