Methotrexate is a folic acid antagonist antitumor drug. A method for the determination of the content and related substances of methotrexate raw materials, providing a reference for the quality standard of methotrexate was established. The instrument was a Thermo Scientific UltiMate 3000 high-performance liquid chromatograph (HPLC), with a Thermo Scientific BDS HypersilTM C18 column (4.0 mm×250 mm, 5 μm); mobile phase: Mobile phase A: (20 mmol/L sodium dihydrogen phosphate, adjusted  pH vaule to 6.0 with NaOH)∶ Acetonitrile=90∶10; Mobile phase B: (20 mmol/L sodium dihydrogen phosphate, adjusted pH vaule to 6.0 with NaOH)∶ Acetonitrile=50∶50; mobile phase A and mobile phase B were eluted according to the elution program; flow rate was 1.0 mL/min; detection wavelength was 280 nm; injection volume was 20 μL; column temperature was 30 ℃. The results showed that the concentration of methotrexate had a good linear relationship in the range of 0.50 ～ 1.50 mg/mL, with the linear equation y = 162.04x + 0.002 (r2 = 0.999); the detection limit was 1.813 μg/mL, and the quantification limit was 6.774 μg/mL; the solution stability indicated that the RSD within 12 hours was 0.25%; the repeatability RSD was 0.18% (n = 6); the intermediate precision RSD was 0.19% (n = 12); the average recovery rate of the added sample was 99.50% (RSD was 0.55%); the content of five batches of methotrexate raw materials was all within 98.0% ～ 102.0%. The results indicated that the method had strong specificity, good reproducibility and solution stability, and the intermediate precision and recovery rate met the requirements, and could be used to detect the content of methotrexate raw materials.