The interest in the development of methods to reduce greenhouse gases has increased enormously. Therefore, one focus is on the production of biofuels like advanced generation bioethanol from lignocellulosic agricultural residues such as wheat straw. The main focus of this work is the specific separation of hemicellulose into soluble xylose and its oligomers from pre-treated wheat straw in order to generate an additional carbon source for microorganisms. Wheat straw was pre-treated by either steam explosion, acid or alkaline pre-treatment and the amounts of xylose that were generated were compared. The best method for hemicellulose separation was the acid pre-treatment.