In times of energy and oil crisis new ways of producing liquid biofuels are evaluated. Bioethanol from lignocellulosics is one of those biofuels that is predicted to be used as a renewable transportation energy source. The major steps for bioconversion of lignocellulosics into bioethanol are pretreatment, enzymatic hydrolysis and fermentation. The enzymatic hydrolysis is a crucial but still costly step. To decrease the direct costs of this process, means to reuse the robust enzymes in subsequent hydrolysis reactions are under investigation. Strong adsorption onto the pretreated substrate for hydrolysis and then release back to the liquid phase is highly advantageous in many recycling processes. Hybrid poplar (Populus nigra x P. maximowiczii) and mountain pine beetle (Dendroctonus ponderosae) killed Lodgepole pine (Pinus contorta) chips were pretreated at various conditions using an ethanol organosolv process. This library of pretreated substrates were used to determine the influence substrate characteristics have for the ability of a commercial enzyme preparation to adsorb to the substrate and efficiently hydrolyze it without showing non-productive adsorption to the residual substrate. The lignocellulosic substrates produced exhibited different monomeric sugar compositions and also different properties. Substrate properties such as hydrophobicity, pore size, sugar and lignin ratio were correlated against the enzymatic adsorption and hydrolysis, unraveling the most crucial properties responsible for the degree of enzymatic hydrolysis and adsorption. The work presented here will describe chosen methodology used to evaluate those substrate factors. The properties which are important for efficient adsorption, hydrolysis and minimal non-productive adsorption will be presented and highlighted.