At present, the world's transportation sector is being principally supplied by fossil fuels. However, energy consumption in this sector is drastically increasing and there are concerns with supply, cost, and environmental issues with the continuing use of fossil fuels. Utilizing non-petroleum ethanol in the transportation sector reduces the dependence on oil, and allows for cleaner burning of gasoline. Lignocellulose materials are structurally composed of five types of polymeric sugars, glucan, galactan, mannan, arabinan, and xylan. NREL has developed a quantitative saccharification (QS) method for determining carbohydrate composition. We proposed a new protocol based on the NREL 2006 Laboratory Analytical Procedure "Determination of Structural Carbohydrates and Lignin in Biomass" (Sluiter et al. 2006a) with a slight modification, in which xylose concentration was determined after the secondary hydrolysis by using 1% sulfuric acid rather than 4% sulfuric acid. We found that the current NREL protocol led to a statistically significant overestimation of acid-labile xylan content ranging from 4 to 8 percent. Lignocellulosic biomass is naturally recalcitrant to enzymatic hydrolysis, and must be pretreated before it can be effectively used for bioethanol production. One such pretreatment is a fractionation process that separates lignin and hemicellulose from the cellulose and converts crystalline cellulose microfibrils to amorphous cellulose. Here we evaluated the feasibility of lignocellulose fractionation applicable to the hurds of industrial hemp. Hurds are the remaining material of the stalk after all leaves, seeds, and fiber have been stripped from the plant. After optimizing acid concentration, reaction time and temperature, the pretreated cellulosic samples were hydrolyzed to more than 96% after 24 hours of hydrolysis (enzyme loading conditions of 15 FPU/g glucan Spezyme CP and 60 IU/g glucan Novozyme 188) at the optimal pretreatment condition (> 84% H₃PO₄, > 50 °C and > 1 hour). The overall glucose and xylose yields were 89% (94% pretreatment; 96% digestibility) and 61%, respectively. All data suggest the technical feasibility of building a biorefinery based on the hurds of industrial hemp as a feedstock and a new lignocellulose fractionation technology for producing cellulosic ethanol. The choice of feedstock and processing technology gives high sugar yields, low processing costs, low cost feedstock, and low capital investment. / Master of Science