The HPLC method used for determination of mitoxantrone was validated by linearity, sensitivity, precision, accuracy and specificity. Retention time for mitoxantrone was 4.3 min and that for haloperidol (internal standard) 7.9 min. Calibration curves were constructed for mitoxantrone over the concentration range 0.25-5 μg.mL-1 (haloperidol, 2 μg.mL-1). For six replicate samples at 0.5, 2, 4, 5 μg.mL-1, intra-day precision values were within 1.63%. Inter-day precision values at the same concentrations were within 1.90%. The limit of detection of mitoxantrone was 0. 1 μg.mL-1. The method was proved to be applicable to the determination of mitoxantrone.