Abstract: OBJECTIVE To establish an HPLC method for simultaneous determination of related substances and content of hydroxycamptothecin injection. METHODS The chromatographic column was Ultimate XB-C18(4.6 mm×250 mm, 5 μm), and the mobile phase consisted of a mixture of methanol-water(60∶40). The flow rate was 1.0 mL·min^-1 and the detection wavelength was 370 nm. The column temperature was maintained at 35 ℃。RESULTS For the determination of related substances: the separations between hydroxycamptothecin and its related substances were very well. The calibration curves of hydroxycamptothecin and its main related substance, namely camptothecinthe, were both linear within the ranges of 0.2-1.0 μg·mL^-1(r≥0.999 7), and the LODs were 0.2 ng and 0.3 ng, respectively. For the determination of content: hydroxycamptothecin was linear within the range of 4.0-36.3 μg·mL^-1(r=1.000 0), the average recoveries of low, medium and high concentrations based on the formulations from manufactory A, B and C were between 100.0% and 102.0%, the RSDs were between 0.02% and 0.71%(n=3). CONCLUSION The method is specific and accurate, and can systematically be used for the quality control of hydroxycamptothecin injection.