Technical Abstract: Starch has unique physicochemical characteristics among carbohydrates. Most starch granules are a mixture of two sugar polymers: a highly branched polysaccharide named amylopectin, and a basically linear polysaccharide named amylose. The objective of this study was to develop a simple, one-step and accurate method for simultaneous determination of amylose:amylopectin ratio as well as weight-averaged molecular weights of starch in whole meal cereal samples. Starch from samples of spring wheat, barley, oat, rye, buckwheat, rice, durum wheat and corn were extracted using KOH, urea and ethanol. Starch samples were solubilized before they were run on the high performance size exclusion chromatography (HPSEC) system with a refractive index detector. To verify the identity of the peaks, fractions were collected and then soluble starch and beta-glucan assays were performed in addition to gas chromatography analysis. All the fractions contained only glucose, which indicated there was no contamination from non-starch polysaccharides. Soluble starch assays matched with the HPSEC profiles. Weight-averaged molecular weights were calculated using a series of gel permeation chromatography grade dextrans. The method described in this study can be used to determine amylose:amypectin ratio and weight-averaged molecular weight of cereal starches using as low as 25mg whole meal samples.