A high-performance liquid chromatography coupled with mass spectrometry (HPLC–MS) method was established for the separation and determination of acetyl-glutamine enantiomers (acetyl-L-glutamine and acetyl-D-glutamine) simultaneously. Baseline separation was achieved on Chiralpak AD-H column (250 mm × 4.6 mm, 5 μm). n-Hexane (containing 0.1% acetic acid) and ethanol (75:25, v/v) were used as mobile phase at a flow rate of 0.6 mL/min. The detection was operated in the negative ion mode with an ESI source. [M-H]? m/z 187.0540 for enantiomers and [M-H]? m/z 179.0240 for aspirin (IS) were selected as detecting ions. The linear range of the calibration curve for each enantiomer was 0.05–40 μg/mL. The precision of this method at concentrations of 0.5–20 μg/mL was within 7.23%, and the accuracy was 99.81%–107.81%. The precision at LOQ (0.05 μg/mL) was between 16.28% and 17.56%, which was poor than that at QC levels. The average extraction recovery was higher than 85% for both enantiomers at QC levels. The pharmacokinetics of enantiomers was found to be stereoselective. There was not chiral inversion in vivo or in vitro between enantiomers.