OBJECTIVE： To extablish the method for blood concentration determination of mitoxantrone in rats， and to study the pharamokinetics of mitoxantrone in rats. METHODS： Totally 6 SD rats were collected and given mitoxantrone 5 mg/kg via tail vein. The blood samples 0.3 mL were collected before medication and 5， 10， 20， 40， 60， 120， 240， 480， 720 min after medication. Blood samples were placed in heparinized EP tube， and the plasma was centrifuged and separated. After adding silica gel， the plasma were ground and mixed well， then added into methanol solution containing 0.5 mol/L hydrochloric acid to precipitate protein. After grinding and mixing， the supernatant was centrifuged and dried with nitrogen and then dissolved with mobile phase. HPLC method was adopted to determine the plasma concentration of mitoxantrone. The determination was performed on ZORBAX SB-C18 column with mobile phase consisted of 20 mmol/L ammonium acetate （pH adjusted to 2.0 with hydrochloric acid）-methanol （65 ∶ 35， V/V） at the flow rate of 1.0 mL/min. The detection wavelength was set at 244 nm， and column temperature was 30 ℃. The sample size was 20 μL. Pharmacokinetic parameters were calculated with DAS 3.0 software. RESULTS： The linear range of mitoxantrone were 200-10 000 μg/L （r＝0.999 6， n＝6）. The lower limit of quantitation was 200   μg/L， and the limit of detection was 150 μg/L， respectively. RSDs of intra-day and inter-day precision and stability were all lower than 8.0％ （n＝5， 3， 6， respectively）. The extraction recoveries were （85.64±3.93）％-（92.31±1.68）％ （n＝3）. The recoveries of accuracy test were （93.58±1.42）％-（113.92±2.74）％ （n＝3）. The pharmacokinetic parameters of mitoxantrone were as follows as AUC0-720 min was （5 247.1±474.6.0） μg·h/L； t1 /2z was （24.88±6.94） h； CLZ was （0.46±0.09） L/（h·kg）； Vz was （11.07±2.64） L/kg. CONCLUSIONS： The method has recovery and good repeatability， and is suitable for the determination of blood concentration of mitoxantrone and its pharmacokinetic research.