Lignocellulosic biomass has long been recognized as a potential low-cost source of mixed sugars for fermentation to fuel ethanol. The low rate at which biomass is converted to sugars and the coproduction of fermentation inhibitors result in pretreatment strategies to prepare the biomass for subsequent biological conversion to biofuel. New approaches for designing improved energy feedstocks, deconstructing plant cell walls, and transforming their polysaccharides to fermentable sugars are needed. Distillers dried grains with solubles (DDGS), the residue from current ethanol production, and switchgrass were selected as biomass feedstocks. Heating was by way of convection and the biomass samples were hydrolyzed at 140°C for 20 minutes with a mixture of dilute sulfuric acid, ethanol and methyl isobutyl ketone, mimicking the NREL (National Renewable Energy Laboratory) clean fractionation procedure. The resulting extracts comprised an aqueous layer and an organic layer. The layers were separated and analyzed with HPLC for determination of sugars, byproducts and degradation products. The biomass residues were analyzed for determination of cellulose, hemicellulose and lignin following the procedures patterned after the NREL standard analytical procedures and modified for biomaterial process streams. The aqueous layer consisted mostly of hemicellulose; the organic phase comprised lignin and the cellulose remained in the biomass residues. Mannose was the most abundant sugars in all layers, whereas glucose was the least represented sugars in all layers. Continuing studies are investigating the presence of single sugars, cellulose; hemicellulose, and lignin in biomass residues.