A sensitive assay was developed for the quantitation of vinblastine, desacetylvinblastine and vincristine using liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC-APCI-MS). Analyses were performed on an Ultrasphere C18 microbore column using ammonium acetate as mobile phase. The calibration curves were linear across the range of 0.51-4.00 ng/ml (0.63-4.93 nM) for vinblastine, 0.74-3.93 ng/ml (0.96-5.11 nM) for desacetylvinblastine and 0.30-3.95 ng/ml (0.36-4.79 nM) for vincristine. Vinca alkaloid concentrations were measured with an accuracy and precision within 11%. This assay could be implemented to determine the plasma concentrations for pharmacokinetic studies of vinblastine, desacetylvinblastine and vincristine in conjunction with clinical trials.