A sensitive, simple and rapid high performance liquid chromatographic-tandem mass spectrometric (HPLC-MS/MS) method was developed for simultaneous quantitation of epothilone D and its major metabolite, the hydrolytic metabolite, epothilone C as internal standard in human plasma. Plasma samples were precipitated with acetonitrile, the analysis used a Venusil ASB C(18) analytical column. A tandem mass spectrometer equipped with electrospray ionization source was used as detector and operated in the positive-ion mode. Selected reaction monitoring (SRM) using the precursor to product ion pairs of m/z 492.3-->304.1 (epothilone D), m/z 510.3-->492.3 (metabolite), m/z 478.3-->290.1 (internal standard) was used for quantification. The analytical method was validated in terms of specificity, precision, accuracy, extraction recovery, stability, matrix effect and dilution effect. The linear calibration curves of epothilone D and metabolite were obtained over the concentration range of 0.2-1000 ng/ml and 5.0-1000 ng/ml, respectively. Lower limits of quantification (LLOQ) of epothilone D and metabolite were 0.2 ng/ml and 5.0 ng/ml, respectively. Due to its high sensitivity, specificity and simplicity, the method could be used for pharmacokinetic studies of both epothilone D and its hydrolytic metabolite.