Sieving methods have been almost exclusively used for feedstock size-reduction characterization in the biomass refining literature. This study demonstrates a methodology to properly characterize specific surface of biomass substrates through two dimensional measurement of each fiber of the substrate using a wet imaging technique. The methodology provides more information than sieving methods about biomass substrate. The measured dimensions of individual fibers were used to estimate the substrate external surface based on a cylinder model. The substrate specific surface and mechanical milling energy consumption were then correlated to enzymatic hydrolysis glucose yield. Results indicated that the developed methodology is effective in differentiating various size-reduction and chemical pretreatment processes in terms of cellulose to glucose conversion efficiency and size-reduction energy consumption. Thermomechanical disk milling (DM-I), exposing cellulose, is more effective than a high pressure thermomechanical disk milling (DM-II), exposing lignin, in subsequent enzymatic hydrolysis. However, DM-I is more energy intensive than DM-II. Both DMs that produce fibers are more efficient in enzymatic hydrolysis than hammer milling that produces fiber bundles. Chemical pretreatment not only increased cellulose conversion, but also reduced mechanical milling energy consumption. The present methodology identified the sulfite pretreatment C as the most efficient pretreatment in terms of glucose yield and milling energy consumption. Published by Elsevier Ltd.