OBJECTIVE： To establish a method for the determination of related substances in Methylphenidate hydrochloride for injection. METHODS： HPLC method was adopted. The determination was performed on Waters symmetry C18 column with mobile phase consisted of methanol-0.01 mol/L potassium dihydrogen phosphate solution （60 ∶ 40，V/V） at the flow rate of 1.0 mL/min. The detection wavelength was set at 210 nm， the column temperature was 35 ℃ and sample size was 10 μL. RESULTS： The linear range of impurity A and B were 0.02-3.0 μg/mL （r＝0.999 8）. The limits of quantitation were 0.2， 0.6 ng， and the limits of detection were 0.06， 0.2 ng， respectively. RSDs of precision， stability and reproducibility were all lower than 2.0％； recoveries were 98.2％-100.0％ （RSD＝0.56％，n＝9）， 98.0％-100.3％ （RSD＝0.70％，n＝9）， respectively. CONCLUSIONS： The method is simple， accurate and suitable for the determination of related substance in Methylphenidate hydrochloride for injection.