A novel fractionation method of corn stover was developed to produce three major product streams, such as cellulose, hemicellulose, and lignin. This process consists of two step hydrolysis; (1) it separates hemicellulose sugars in the first stage using ZnCl₂, followed by (2) lignin separation in the second stage using ammonium hydroxide. In this method, ZnCl₂ was used to hydrolyze the hemicellulose into xylooligomer in the first stage and lignin hydrolysis using ammonium hydroxide was operated in succession. The remaining solid thus contains mostly cellulose. We found that ZnCl₂ has high selectivity for hemicellulose hydrolysis (70-80%), while it retained most of lignin and cellulose (>97%) with the solid. In addition, delignification method of biomass using ammonium hydroxide has been developed in our laboratory; we have reported it is effective for lignin removal. When this fractionation process is properly operated, near complete fractionation is therefore achieved. Each of separated components can be used for the production of value added chemicals using either biological or chemical conversions since they are recovered in highly pure form. Remaining cellulose after fractionation is relatively pure and highly digestible to commercial cellulase enzymes because fractionation process removes lignin and hemicellulose in the earlier stages. The effects of process conditions on the compositional changes of corn stover were tested. The enzymatic digestibility tests were conducted for the corn stover treated under various conditions. Other technical aspects pertinent to development of fractionation process will be presented.