HPLC method for determination of ajoene isomers in garlic oil products was optimized and validated. E- and Z-Ajoene were extracted with ethyl acetate and followed by the sensitive and selective determination of 2 isomers in a single run using normal phase HPLC equipped with silica gel column. The mobile phase was n-hexane and 2-propanol (85/15, v/v) with an isocratic condition as a flow rate of 1.0 mL/min and 240 nm of HPLC UV detector. All calibration curves of E- and Z-ajoene in oil-macerated garlic showed good linearity (r=0.998). Overall, intra- and inter-day were in the range of 0.12-2.30 and 2.84-5.26%, respectively. Recovery was in range of 87.17-98.53% for E-ajoene and 85.16-99.23% for Z-ajoene. The validated method was applied to determine contents of ajoene in macerate garlic juices prepared with various vegetable oil. There were no any matrix effects in all chromatograms. The proposed method may be useful for quality control and evaluation of garlic oil products.