Objective To establish a HPLC-MS/MS method to si-multaneous determination free doxorubicin, and five metabolites(M1-M5). Methods Daunorubicin was selected as internal standard After plasma samples were treated with solid phase extraction, chromatog. separation was achieved on a ZORBAX Extend-C_(18)(150 mmx 4.6 mm, 5 μm) column through gradient elution by 0.1% formic acid with acetonitrile. The analytes were detected by API 4000 mass spectrometer with the electrospray ionization(ESI) in multiple reaction monitoring(MRM) mode. Doxorubicin and five metabolites were detected using pos. ion mode. The precursor/product ion transitions of m/z 544.3→m/z 397.1(doxorubicin), m/z 545.9→m/z 399.1(M1), m/z 397.0→m/z 361.0(M2), m/z 399.2→m/z 363.0(M3), m/z 399.2→m/z 381.1(M4), m/z 401.2→m/z 297.0(M5) and m/z 528.3→m/z 321.1(daunorubicin). Results The quant. linear ranges were 5-5000 ng·mL∼(-1) for doxorubicin and 5-1500 ng·mL∼(-1) for its five metabolites in rat plasma. Intra-day and inter-day precision for quality control samples of doxorubicin and five metabolites were good. Extraction recovery and matrix effects were in line with requirements. Conclusion This method is simple, rapid, sensitive and suitable for the pharmacokinetic studies of doxorubicin hydrochloride preparation