New requirements for the biofuels industry force individual enterprises to develop various procedures for newly selected substrates pretreatments that could be applicable in processing of large quantities of raw materials. Even greater pressures are on second-generation biofuels producers justified by selection of waste lignocellulosic substrates and methods of substrate processing. Among the most suitable lignocellulosic raw materials in Slovak Republic (SR) for 2G bioethanol production is wheat straw. This raw material (Senec region, SR) for enzymatic hydrolysis was pretreated by dry milling (Brabender), cyclic freezing and thawing, wet milling (Sprout Waldron), two-step process of steam explosion at 180°C and extrusion at 145°C and one-step process of steam explosion at different temperatures. Wheat straw holocellulose accessibility was tested by adsorption of three commercially available dyes (Pylam Products Company, Inc., USA). Absorptivity coefficient of each dye at its maximum wavelength was determined from individual calibration curves of dyes and their values resulted ranging from 13.78 to 19.52 dm3.g-1.cm-1. The absorption of solution was measured and concentration of residual dye was calculated at given wavelength. The accessibility of holocellulose contained in wheat straw pretreated by steam explosion was controlled by SEM (scanning electron microscope) in correlation with the ratio of adsorbed dyes according to the modified Simons’ method.