Overcoming the natural recalcitrance of biomass to microbial breakdown is essential to any operation that converts biomass to fermentable sugars. Pretreatments provide the key to unlocking the protective structures in biological feedstocks so that enzymatic hydrolysis of the carbohydrate fraction to monomeric sugars can be achieved more rapidly and with greater yield. Numerous pretreatment concepts have been proposed and tested over the years. Steam treatment (or steam explosion) with and without the addition of a catalyst is one of the oldest methods. A coordinated study supported by a USDA Initiative for Future Agricultural and Food Systems (IFAFS) grant has examined, in phase I of the project, the performance of five promising biomass pretreatment methods. Almost all the five pretreatment methods, i.e., dilute acid, hot water (neutral pH), ammonia fiber/freeze explosion (AFEX), ammonia recycle percolation (ARP), and lime can produce high sugar yield. Nevertheless, it is found that low cost pretreatment reactors are often counterbalanced by the higher costs associated with either pretreatment catalyst recovery or higher costs for ethanol product recovery. Although a number of technically feasible approaches for biofuel production from lignocellulosic biomass have been explored, the development of economically feasible biofuel production methods remains a key challenge. Up to 67% of the total cost in cellulosic ethanol production is the processing costs and pretreatment is the most expensive single unit operation. While researchers are scrutinizing each step in biomass conversion to minimize the production costs, the concept of biorefinery puts effort on enhancing the cost structure of biofuel generation. Similar to its successful counterpart in the petrochemical industry, the ultimate goal of a biorefinery is the efficient fractionation of lignocellulosic biomass into multiple streams that contain value-added compounds so that the overall economics of a biofuel production facility can be significantly improved. A two-stage concept was used in this study to fractionate Miscanthus into valued-added products (hemicelluloses and lignin) and fermentable sugars under relatively mild condition. Using catalysts such as acidic and alkaline electrolyzed water, dilute sulfuric acid, or alkaline hydrogen peroxide, the digestibility of Miscanthus by a one-step pretreatment at 160, 170, 180, 190, 200, or 210 oC for 8, 16, or 24 min was compared with a two-stage fractionation at 50 and 121 oC. FTIR was used to characterize structure changes in the pretreated samples.