Background: The development of the second generation ethanol production technology requires new, highly effective methods of pretreatment of lignocellulosic biomass, which reduces lignin content, eliminates fermentation inhibitors, and makes the biomass susceptible to hydrolysis using cellulolytic enzymes. New pretreatment methods should be adapted to the processing of lignocellulosic waste biomass from various industries. One of the problems is the management of grain stillage, which is waste from the production of first generation ethanol.Results: The aim of the study was to assess the suitability of the combined use of microwave radiation and sodium cumene sulfonate under optimized process conditions for the preparation of maize stillage biomass as a raw material for the production of cellulosic ethanol. The key parameter guaranteeing a high level of lignin removal from biomass (ca. 44%) was the concentration of hydrotrope. The highest organic matter extraction from biomass (67.00±1.68%) was observed for 20% v/v sodium cumene sulfonate, ca. 117 PSI (microwave heating) and 30 min exposure time. Even at high biomass concentration (16% w/v) and a cellulose enzyme dose of about 4 FPU/g, maize stillage biomass subjected to microwave-assisted hydrotropic pretreatment was highly susceptible to enzymatic degradation, which resulted in 80% hydrolysis yield. The stillage biomass processed in this way is a very good raw material for the production of cellulosic ethanol. It is possible to obtain a fermentation medium with a very high glucose concentration (up to 80 g/L), without fermentation inhibitors (such as 5-HMF, furfural, lignin degradation products) and, as a consequence, to reach a very high level of sugar conversion to ethanol, even as much as 95% of theoretical yield.Conclusions: Microwave hydrotropic treatment with sodium cumene sulfonate is a very effective way to prepare waste maize stillage biomass for the production of cellulosic ethanol. The method provides a high level of enzymatic degradation of cellulose, leading to a medium with high content of released sugars suitable for bioconversion, which is in line with the assumptions of the second generation ethanol production technology.