An assay for the quantitative determination of docetaxel in human plasma is described. Docetaxel was extracted from the matrix using liquid–liquid extraction with ter-butylmethylether, followed by high-performance liquid chromatographic analysis using an alkaline eluent. Paclitaxel was used as internal standard. Positive ionization electrospray tandem mass spectrometry was performed for selective and sensitive detection. The method was validated according to the FDA guidelines on bioanalytical method validation. The validated range for docetaxel was from 0.25–1000 ng/mL using 200 µL plasma aliquots. The method requires only a limited volume (200 µL) of human plasma and the method can be applied in studies requiring a low lower limit of quantitation of 0.25 ng/mL. The assay was applied successfully in several clinical and pharmacological studies with docetaxel. Copyright © 2004 John Wiley & Sons, Ltd.