Abstract: A high performance liquid chromatographic method for determining starch in tobacco with an internal standard (IS) was developed.The starch in tobacco was hydrolyzed into glucose with hydrochloric acid.Then the glucose content was determined on a Waters Sugar-Pak 1 cation exchange column with 0.05 g/L EDTA calcium disodium salt solution as mobile phase and detected with a differential refractive index detector using mannitol as an IS. The limit of detection was 1.0 mg/L for glucose.The recoveries of the method were from 95% to 105%, and the relative standard deviations of the method were in the range of 1.6%-2.1%.The starch in several tobacco samples was determined with the method.