Abstract: The method for the determination of lactic acid enantiomers in Massa Medicata Fermentata by high performance liquid chromatography (HPLC) was established, and the sample extraction method was optimized. The sample was crushed and extracted using ultrasonic extraction with purified water, centrifuged at 10 000 r/min for 5 min. The supernatant was filtered using a filter membrane, eluted with 2 mmol/L CuSO₄ solution (5% isopropyl alcohol as solvent) as mobile phase at a flow rate of 0.60 mL/min, separated chirally on a Chirex 3126 (D)-penicillami (4.6 mm×250 mm, 5 μm) column, and detected using a diode array detector, quantified by external standard method. The results showed that L-lactic acid and D-lactic acid had good linearities in their ranges (r ≥ 0.999 7), and the average recoveries were 98.5% and 97.1%, respectively. The relative standard deviations (RSD) were 1.8% and 2.4%, respectively. The method is simple, feasible, stable, and reliable, and can provide a reference for the quality improvement of Massa Medicata Fermentata.