A rapid, simple and sensitive high-performance liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) method for the quantification of vinflunine in rat plasma was developed and validated. After making alkaline with NaOH, plasma was extracted with ethyl acetate and determined by LC-MS. The analysis was carried out on a Shimadzu VP-ODS column (150 mmx4.6 mm ID, packed with 5 microm C18 Silica RP particle). The mobile phase consisted of methanol-10 mM ammonium acetate buffer (80:20, v/v) with the flow rate of 1.0 ml/min. LC-MS was performed in the selected ion monitoring (SIM) mode using target ions at m/z: 817.3 for vinflunine and m/z: 373.2 for finasteride (IS). Chromatographic separation was achieved in less than 6 min and the calibration curve was linear over a concentration range of 0.025-6.25 microg/ml. The intra-assay and inter-assay variability values were less than 8.6%. The accuracy ranged from 91.5 to 105.6%. The established method has been successfully applied to a pharmacokinetic study of vinflunine in rats.