A simple, fast and economical HPLC assay for the determination of mitoxantrone in mouse plasma and tissue homogenates is described. Protein precipitation with sequential addition of sulfosalicylic acid and acetonitrile was used for sample preparation. The resolution of mitoxantrone and the I.S. were achieved by using acetonitrile and 10mM sodium phosphate buffer with 0.1% TEA. The separation was performed on a Nucleosil C18, 250mmx4mm I.D. column with UV detection at 610nm. The inter-day and intra-day precision and accuracy of quality control (QC) samples, evaluated both in plasma and tissue homogenates, were all within 15%. The lower limit of quantification (LLOQ) was 5ng/ml in plasma, 25ng/ml in liver homogenate and 12.5ng/ml in other tissue homogenates. This assay was successfully applied in a pharmacokinetic and tissue distribution study of mitoxantrone in mice.