Conversion of lignocellulosic biomass to sugars and ethanol requires an effective pretreatment before the cellulose can be efficiently hydrolyzed by enzymes.  Pretreatment conditions cover the entire range from low to high pH, from moderate to high temperatures, and from minutes to weeks.  As with all chemical processes, pretreatments are only successful if they generate the desired products in high yield and undesirable products are minimized.  Development of tools to understand the effect of pretreatment processes on lignocellulosic feedstocks is an active area of research.  Compositional analysis of the feedstock and product streams provides critical information about the sugar yields.  In acidic pretreatments, high levels of xylan solubilization indicate that the remaining cellulosic solid should be highly digestible by enzymes. Physico-chemical characterization of the lignocellulosic solid product can also indicate pretreatment effectiveness.  Characteristics typically tracked are cellulose crystallinity (by solid-state ¹³C NMR or X-ray diffraction), cellulose accessibility (measured using fluorescence labeled enzymes) and porosity (measured by thermoporometry or solute exclusion).  Various microscopic imaging techniques can be used to follow changes in lignin and xylan distribution in the plant cell wall.  By labeling with carbohydrate specific probes changes in the cell wall structure can be revealed.   Immunoelectron microscopy has been used to monitor how major enzyme components of biomass degrading enzyme cocktails penetrate the cell wall matrix following pretreatment.  Using these tools, we are attempting to gain a better understanding of how pretreatment processes can generate highly digestible cellulosic substrates so that biomass can converted to sugars with the highest possible yields.