Methotrexate (MTX) is an antineoplastic therapeutic medicine as antimetabolite of folic acid. In this paper, a sensitive and rapid ultra-performance liquid chromatographic (UPLC) method was developed and validated for the separation and determination of impurities in MTX drug substances. The UPLC method was accomplished on an Agilent Zorbax Extend C-18 (50 mm×4.6 mm, 1.8 mm) with a gradient elution system composed of sodium dihydrogen phosphate in water (20 mmol/L, pH 3.0) and acetonitrile. The flow rate was 2.2 mL/min. The method was validated. The calibration curves displayed good linearity (r > 0.999) within the tested concentration ranges. The limit of detection (LOD) and limit of quantification (LOQ) of the six analytes were all less than 0.774 μg/mL and 1.03 mg/mL. The relative standard deviation (RSD) for intra-and inter-day precision of the six analytes was less than 9.8%, including at the LOQ. The average recovery ranged from 95.2% to 103% except at the LOQ, where recovery ranged from 82.7% to 117%. The validated method was successfully used to determine the relative abundance of six impurities in the MTX drug substances.