Extrusion processing has shown potential to be used as a pretreatment method for second-generation bioethanol production. Furthermore, surfactants have been shown to reduce enzyme deactivation and increase the efficiency of hydrolysis. Therefore, a sequential pretreatment technique was developed for corn stover (CS) and prairie cordgrass (PCG) in which a single screw extruder was used for the first pretreatment according to a previously optimized condition using 70-180 °C for feed, barrel, and die zones with 65-155 rpm screw speed. The second pretreatment was optimized in this study at 45-55 °C, 1-4 h, 0.15-0.6 g Tween 20/g glucan according to response surface methodology. Optimization of surfactant pretreatment facilitated the estimation of interaction and higher-order effects for major factors involved in surfactant treatment (temperature, time, surfactant loading). Using 8.6 FPU/g glucan cellulase, the optimum conditions found by fitting appropriate quadratic models to the data increased glucose and xylose yield by 27.5 and 33% for CS and by 21.5 and 27% for PCG, respectively. Tween 20 concentrations and pretreatment temperature were the most significant factors affecting sugar yield (p value <0.05). Studies of SDS concentration at and beyond critical micelle concentration (5.2-100 mM) demonstrated a decrease in sugar yield compared to control.