Abstract: Sample of rhubarb was extracted with mixture of ethanol and water (6+4) by refluxing for 3 times with 0.5 h for each time. The content of total anthraquinones in the extract was determined by spectrophotometry using 1,8-dihydroxy-anthraquinone as standard sample at the wavelength of 428 nm. Under the optimum condition, linear relationship between values of absorbance and mass concentration of 1,8-dihydroxy-anthraquinone was kept in the range of 5.0-25.0 mg·L-1. Test for recovery was made by standard addition method, and values of recovery obtained were in the range of 98.7%-99.8%.