Abstract: A method was developed for the determination of allicin in garlic by HPLC.The sample was extracted with water, and separated on a C18 column with gradient elution with the mobile phase of acetonitrile and water. The retention time was used for qualitative analysis.The external standard calibration curves were used for quantitative analysis.The detection wavelength was 245 nm. The calibration curve was linear between the peak area and the mass concentration of allicin from 6.25 to 500 mg / L with the correlation coefficient of 0.9999.Repeatability and reproducibility of the method were validation by four laboratories, for all the samples, the mean spiked recoveries of allicin were in the range from 89.0% to 107%, and the relative standard deviations ( RSDs, n = 3) were in the range of 1.39% ⁶.62%. The repeatability standard deviation was less than the repeatability limit and the reproducibility standard deviation was less than the reproducibility limit. The method was simple, accurate and feasible for determination of allicin in garlic.