Parameters such as pretreatment method, enzyme type and concentration, determine the conversion efficiency of biomass’ cellulose and hemicellulose to glucose and mainly xylose in biomass-based fuel production. Chemical quantification of these processes offers no information on the effect of enzymatic hydrolysis (EH) on particle morphology. We report on the development of a microscopy method for imaging pretreated biomass particles at different EH stages. The method was based on acquiring large field of view images, typically 20×10 mm2 containing thousands of particles. Morphology of particles with lengths between 2 μm and 5 mm could be visualized and analyzed. The particle length distribution of corn stover samples, pretreated with increasing amounts of sulfuric acid at different EH stages, was measured. Particle size was shown to be dependent on pretreatment severity and EH time. The methodology developed could offer an alternative method for characterization of EH of biomass for second generation biofuels and visualization of recalcitrant structures.