Pretreatment of plant biomass is a key stage in the production of second-generation biofuels, as it ensures the destruction of the lignocellulosic structure and increases the availability of polysaccharides for further conversion. The paper investigates the effectiveness of various methods of processing oat straw for the isolation of the polysaccharide component. Mechanical, aqueous, alkaline and organic methods, as well as combined organosolvent processing using acetic acid and hydrogen peroxide, are considered. It was found that the highest yield of solid product is provided by processing with ethanol (98.7 %) and alcohol-benzene mixture (96.8 %), however, the obtained materials retain the original structure of biomass. Instead, hot water (82.7 %) and alkali (58.0 %) lead to partial extraction of soluble substances and hemicelluloses, while the use of peracetic acid gives the lowest yield (53.1 %), but provides deep delignification, ash reduction and formation of a porous structure suitable for enzymatic hydrolysis. Thus, it is the peroxide-acetic acid treatment that has proven to be the most promising for increasing cellulose availability and preparing oat straw for further biotechnological use in biofuel production.