OBJECTIVE：To establish a method for the content determination of Vincristine sulfate for injection. METHODS： HPLC was performed on the column of Agilent HC-C8 with mobile phase of 1.67％ Diethylamine aqueous solution（adjusted to pH 7.5 with phosphoric acid）-methanol（38 ∶ 62，V/V）at a flow rate of 1.0 ml/min，the detection wavelength was 297 nm，the column temperature was 35 ℃，and the injection volume was 10 μl. RESULTS：The linear range of vincristine sulfate was 0.032-3.2 mg/ml （r＝0.999 9）；RSDs of precision，stability and reproducibility tests were lower than 1％；recovery was 99.3％-101.7％（RSD＝ 0.6％，n＝9）. CONCLUSIONS：The method is accurate，reliable and sensitive，and can be used for the content determination of Vincristine sulfate for injection.