The use of rice straw (RS) was enhanced to produce biobutanol as biofuel, for which the NaOH pretreatment was optimized by considering the butanol-biomass ratio that quantify the mass balance efficiency of the three sequential stages of the process: pretreatment, enzymatic hydrolysis and fermentation by Clostridium beijerinckii. The optimum point (solid loading of 5% w/v with 0.75% w/v NaOH at 134 °C for 20 min) of the best cost-wise option yielded an enhanced biomass use of 77.6 g kg RS−1. A maximum butanol titer of 10.1 g L−1 was reached after 72 h of fermentation with the complete uptake of glucose and nearly complete uptake of xylose. The NaOH concentration was the most influential parameter. The appropriate dosage to maximize fermentable sugars instead of the mass balance efficiency of the three stages underestimated the biomass use by 13%, showing the importance of correctly selecting the variable response during optimization.