AbstractDifferent substrate characteristic analyses have been studied on rice and triticale straw pretreated with NMMO (N-methylmorpholine-N-oxide) prior to biogas production. Simons’ stain, water retention value (WRV), and enzymatic adsorption were used to measure the change in the accessible surface area of the lignocellulosic substrates. FTIR was used to measure the change in cellulosic crystallinity and Time-of-Flight-Secondary-Ion-Spectroscopy (ToF-SIMS) to measure the ratio of cellulose to lignin on the sample surface. All methods showed increased accessible surface area and a decrease in crystallinity after the pretreatments. These qualities were linked to improved biogas production. In the future, the tested methods could replace the time-consuming methane potential analysis to predict the methane production of lignocellulosic materials. Simons’ stain, enzymatic adsorption, and crystallinity measurement by FTIR can be regarded as the recommended methods for the prediction of the improved biogas production as a result of the pretreatment.