Abstract: A reversed phase high performance liquid chromatography(RP-HPLC)method for the determination of phenyllactic acid(PLA)in fermentation broth of lactic acid bacteria(LAB)was established and optimized.The effects of three stationary phases,two organic phases,five water phases and their proportions on the determination of PLA by HPLC were investigated. The double capped C₁₈ column(4.6 mm×250 mm,5 μm)was selected as the stationary phase,isoelution with 0.05% TFA acetonitrile(75∶25,v/v).The flow rate was 1 mL/min,the detection wavelength was 210 nm,and the column temperature was 30 ℃. The results showed that the linear range of the calibration curve for PLA was 0.2~5.0 mg/L with a correlation coefficient of 0.9998. The recovery rate was 99.88%~101.08%,LOD was 0.1400 mg/L,LQD was 0.4600 mg/L,relative standard deviation(RSD)of precision and repeatability were 0.30% and 0.64% in 1 d,precision and repeatability RSD were 0.59% and 0.79% in 30 d,stability of PLA standard stock solution RSD was 2.45%. Under the 95% confidence interval(coverage factor k=2),when the amount of PLA in the fermentation broth was 0.1400,the expanded uncertainty was 0.0046 mg/L. The yield of PLA synthesizd by Lactobacillus brevis P3 and Lactobacillus plantarum ATCC8014 were 77.58 and 62.60 mg/L after 120 h of culture,respectively. The results demonstrate that PLA can be detected stably,with high efficiency,low cost,simple operation,accurate and reliable results under the proposed method.