A useful and rapid method for the analysis of mitoxantrone in human plasma is described. We purified the sample with a C18 Sep Pak cartridge pre-treated with methanol, water and 0.05 M ammonium phosphate (pH = 2.75). The drug and internal standard (Methylene Blue) were eluted from the cartridge with 1 M acetic acid in methanol and were separated on a 10 microns particle size CN Resolve cartridge in conjunction with a radial compression liquid chromatograph. The mobile phase consisted of a mixture of 0.05 M ammonium phosphate, acetonitrile and methanol (60:35:5, by vol), and the detection was performed spectrophotometrically at 660 nm. The peak height ratio (drug/internal standard) varied linearly (r greater than 0.993) with concentration over the range examined 0.01-3 micrograms ml-1, and the inter- and intra-run precision at high, medium and low concentrations were good; CV ranged from 2.52 to 7.2%. There was no interference from other anticancer drugs or analgesics. We applied the described method to investigate the pharmacokinetics of mitoxantrone using a rabbit as an in vivo model.