A quantitative analysis of alisol compounds in Alismatis Rhizoma (AR) using HPLC was developed. Three alisol bioactive compounds were determined. The separation was optimized using Optimapak C18 column (250 x 4.6 mm, 5 um) with mobile phase of acetonitrile and 0.1 percent aqueous phosphoric acid and detection wavelength of 205 nm. The validation gave acceptable linearities (r 0.9997) and recoveries (98.06 percent to 101.71 percent). The limit of detection (LOD) of the compounds was ranged from 0.08 to 0.15 ug/ml. Contents of alisol C acetate, alisol Band alisol B acetate in AR were found to be 0.05 - 0.76, 0.53 - 5.99, and 3.43 - 11.61 mg/g dried weight, respectively.