L-Se-methylselenocysteine is a new selenium-containing food supplement; however, no studies regarding the safety of D-Se-methylselenocysteine have been reported. Current methods for the determination of L-Se-methylselenocysteine content mainly focus on the measurement of total Se-methylselenocysteine content and cannot differentiate it from the content of L-Se-methylselenocysteine. High-pressure liquid chromatography (HPLC) was used in this study to estimate L-Se-methylselenocysteine content using chiral separation, and a standard curve was established. Reliable chromatographic separation was achieved on an EC250/4 NUCLEOSIL Chiral-1 column (4 mm × 250 mm, 5 μm) when the mobile phase was 0.4 mmol/L CuSO4?5H2O, column temperature was 35 ℃, flow rate was 1.0 mL/min, sample size was 20 μL, and detection wavelength was 240 nm. Under these conditions, the calibration curves established showed good linearity across the concentration range of 0.541~1.352 mg/ml, with a correlation coefficient of 0.9999 and an average recovery of 97.73%. The method is simple, accurate, and can be used to monitor the quality of L-Se-methylselenocysteine and its related products.