Abstract: OBJECTIVE To establish a pre-column derivatization HPLC for determination of L-glutamine in Compound Glutamine Entresoluble capsules.METHODS HPLC experiment was performed on a CAPCELL PAK C₁₈ column (150 mm×4.6 mm, 5 μm). The mobile phase were sodium acetate buffer solution(pH 6.4) and acetonitrile with gradient elution. The detection wavelength was set at 254 nm. Taurine was used as an internal standard substance, the sample and taurine were derivatized with phenyl isothiocyanate. The combination of single factor and orthogonal experiments were used to optimize the derivatization experiment conditions.RESULTS The L-glutamine had good linearity in the range of 0.015 9-0.143 mg·mL^-1(r²=0.999 9). The average recoveries was 100.3% with RSD of 0.8%(n=9).CONCLUSION The HPLC method is accruate with high repeatability, which is helpful for the determination of L-glutamine in Compound Glutamine Entresoluble capsules.