In this work, the effect of hot water pretreatment on glucose production was evaluated. For this purpose, the lignocellulosic structure of sugarcane bagasse and maralfalfa grass, used in this pretreatment, was characterized. It was found that the pretreatment degrades the lignocellulosic structure and increases glucose production after enzymatic hydrolysis. The maximum glucose production for the biomasses pretreated with hot water was 0.984g/L for sugarcane bagasse (10.93min, 9.79% solids, 149.70ºC) and 1.80g/L for grass (17.07min, 16.64% solids, 146.20ºC). These results represent an increase in glucose production of 14.5% and 27.6%, with respect to the samples without pretreatment.