Spectroscopic characterization of both untreated and treated material is being performed in order to determine changes in the biomass and the effects of pretreatment on crystallinity, lignin content, selected chemical bonds, and depolymerization of hemicellulose and lignin. The methods used are X-ray diffraction for determination of cellulose crystallinity (CrI); diffusive reflectance infrared (DRIFT) for changes in C-C and C-O bonds; and fluorescence to determine lignin content. Changes in spectral characteristics and crystallinity are statistically correlated with enzymatic hydrolysis results to identify and better understand the fundamental features of biomass that govern its enzymatic conversion to monomeric sugars. Models of the hydrolysis initial rate and 72 h extent of conversion were developed and evaluated. Results show that the hydrolysis initial rate is most influenced by the cellulose crystallinity, while lignin content most influences the extent of hydrolysis at 72 h. However, it should be noted that in this study only crystallinity, lignin, and selected chemical bonds were used as inputs to the models. The incorporation of additional parameters that affect the hydrolysis, like pore volume and size and surface area accessibility, would improve the predictive capability of the models.