Biofuel production from plant biomass has been proposed as a solution to mitigate fossil fuel use and reduce greenhouse gas emissions. The main components of plant material are structural and non-structural carbohydrates. Structural carbohydrates are bound in the biomass and include cellulose, hemicellulose, lignin and pectins. Non-structural carbohydrates are starches, simple sugars, organic acids, protein, ash, chlorophyll and waxes. Determination of monomeric carbohydrates is important to assess the theoretical ethanol yield. Further, constituent determination of biomass for bioenergy requires the removal of non-structural carbohydrates (extractives) prior to analysis to prevent interference with the analytical procedure. According to the accepted DOE-National Renewable Energy Laboratory method NREL/TP-510-42619, the biomass extractives in corn stover should be removed by a two-step extraction process to remove water and ethanol soluble compounds. The determination of the structural carbohydrates in the pretreated biomass is then performed by the NREL/TP-510-42618 method. This procedure uses a two-step acid hydrolysis which fractionates the biomass polymeric carbohydrates into monomeric forms, which are then analyzed via HPLC. Alternatively, the Neutral Detergent Fiber Analysis (NDF) is a common method used to determine the structural carbohydrate portion of forage biomass. The advantages of the NDF method are its speed as well as the low cost. Using the NDF procedure could allow for theoretical ethanol yield estimation instead of the more resource intensive NREL procedure. The objective of this work is to determine whether the NDF method for determination of structural carbohydrates can be used as an alternative to the NREL procedure.