An integrated flow-through fixed-bed reactor concept for lignocellulose separation at 3 L scale is presented. Wheat straw is degraded using sequential liquid hot water treatment and enzymatic hydrolysis in a single-pot reactor setup. System responses on process parameters are modeled and optimized using response surface methodology. Accurate empiric models and significant factor influences could be identified for both treatment steps. With optimal factor settings, 23.9 and 49.2 wt% hot-water-soluble lignin and C5 oligomers could be recovered, respectively. 70.0 wt% cellulose solubilization could be achieved in the fixed-bed enzymatic hydrolysis after 72 hours.