Enzymatic pre-hydrolysis using the industrial enzymatic cocktail Cellulyve (R) was assessed as a first step in a pretreatment process of Miscanthus biomass involving an aqueous-ethanol organosolv treatment. C-13 and P-31 Nuclear Magnetic Resonance and size exclusion chromatography were used to analyze the cellulose and lignin before and after treatment. It was demonstrated that despite a very low impact on the fibre structure (observed by Scanning Electron Microscopy) and composition (in terms of sugars and polyphenolics content), the enzymatic pre-treatment disrupted the lignocellulosic matrix to a considerable extend. This weakening permitted enhanced removal of lignin during organosolv pulping and increased hydrolysability of the residual cellulosic pulp for the production of monomeric glucose. Using this combined treatment, a delignification yield of 93% and an enzymatic cellulose-to-glucose conversion of 75% were obtained. (C) 2012 Elsevier Ltd. All rights reserved.