Enzymatic hydrolysis is the essential step in the production of 2nd generation biofuels made from lignocellulosic biomass, i.e. agricultural or forestry solid wastes. The enzyme-catalysed robust degradation of cellulose and hemicellulose to monosaccharides requires the synergistic action of the independent types of highly-specific enzymes, usually offered as ready-to-use preparations. The basic aim of the study was to experimentally determine the enzymatic activity of two widely industrially-applied, commercially available cellulolytic enzyme preparations: (i) Cellic® CTec2 and (ii) the mixture of Celluclast® 1.5L and Novozyme 188, in the hydrolysis of pre-treated lignocellulosic biomass, i.e. (a) energetic willow and (b) rye straw, or untreated (c) cellulose paper as well, used as feedstocks. Before the hydrolysis, every kind of utilized lignocellulosic biomass was subjected to alkaline-based (10% NaOH) pre-treatment at high-temperature (121°C) and overpressure (0.1 MPa) conditions. The influence of the type of applied enzymes, as well as their concentration, on the effectiveness of hydrolysis was quantitatively evaluated, and finally the enzyme activities were determined for each of tested cellulolytic enzyme preparations.