Abstract: A high performance liquid chromatographic ( HPLC) method for the determination of Acetyl-CoA in Lactobacillus bulgaricus was developed. The stationary phase used were C18 ( Hypersil ODS2 5μm) chromatographic column and the two mobile-phase solvents used were buffer A (0.2mol/L sodium phosphate, pH5) and buffer B (800mL of 0.25mol/L sodium phosphate mixed with 200mL pH5 of acetonitrile) , gradient elution and at a flow rate of 1mL/min, temperature of column at 25℃, the UV detector was employed to monitor column effluent at 254nm. Results indicated that : Acetyl- CoA and other substances were completely separated and determined in 17min, the linear correlation coefficients were above 0.9995 in the range of0.011 ⁰.359μmol/mL. The limit of detection of acetyl coenzyme A was 0.28mg/L, the limit of quantitation of acetyl coenzyme A was 0.32mg/L. Under these optimized conditions, the recovery of the Acetyl-CoA in Lactobacillus bulgaricus at three spiked levels were in the range of 94.8%¹03.3% with the relative standard deviations (n=6) of 0.75%. This method was fast and accurate for the quantitative analysis of the Acetyl-CoA in Lactobacillus bulgaricus.