Single stage hydrothermal pretreatment was conducted mostly for converting biomass to ethanol. It was optimized to increase the sugar yield benefiting the following ethanol production process, but also produced inhibitors impacting the ethanol yield. Overall optimization must take into account the yield of both the sugars and the inhibitors. We were evaluating two-stage acid-alkaline pretreatment to convert Miscanthus, one potentially profitable biomass in the Midwest, to reduce the production of inhibitors while maintaining a high sugar yield. In the first stage, dilute acid treatment with 0.49% sulfuric acid was conducted at 160°C for 20 min for hemicelluloses removal. The second stage was carried out by aqueous ammonia treatment with 1:1 ammonia biomass loading at 240°C for 10 min for delignification. The results indicated that two-stage pretreatment produced at least 730% more xylose, but 60% less inhibitor (acetic acid) than one-stage pretreatment, two-stage acid or alkaline pretreatment. In addition, one-stage acid pretreatment was optimized to degrade hemicelluloses by varying pretreatment temperature (160, 170, 180°C), residence time (20, 35 min) and sulfuric acid concentration (0.5, 0.75, 1.0 wt%). The highest xylose yield of 12.6% was achieved at 160°C, 20 min, and 0.75 wt%> H 2SO4, with the lowest inhibitor production of 35.8 acetic acid, 0.96 HMF, and 24.1 mg/g dry biomass furfural. Therefore, the advantages of two-stage acid-alkaline pretreatment over the other two-stage pretreatments and one-stage pretreatments was testified and a better operating condition was found through optimization in the acid stage.