Ethanol production from lignocellulosic biomass comprises pretreatment, hydrolysis, and fermentation. However, several inhibitors are generated during rice straw chemical hydrolysis, including furfural, 5-hydroxymethylfurfural (HMF), and phenolics. These inhibitors, i.e., furfural and HMF, are toxic to yeast cells, can negatively impact yeast growth and metabolism, and reduce the process efficiency and production yield. Total phenolics are also reported to inhibit yeast growth and metabolism and act as a source of reactive oxygen species (ROS), which can damage yeast cells. Therefore, minimizing the generation of these inhibitors during rice straw hydrolysis is essential to improve the efficiency and yield of ethanol fermentation. Optimization of process variables can help reduce inhibitor generation and increase the efficiency of used detoxification methods such as adsorption, ion exchange, and biological methods. This study aimed to minimize inhibitor generation during the chemical hydrolysis of rice straw biomass. Minitab 17 software was employed and response surface curve regression analysis was used to develop a quadratic equation of an optimized process for minimized release of inhibitors molecules. The main inhibitors in pretreated rice straw hydrolysate identified were furfural (48.60%/100 g solid biomass), HMF (2.32%/100 g solid biomass), and total phenolics (1.65%/100 g solid biomass). The optimal pretreatment conditions were a biomass solid loading rate of 15% w/v, an H2SO4concentration of 12% v/v, a pretreatment reaction time of 30 min, and a temperature of 100 °C. Optimization of these process variables reduced the inhibitor generation by up to one and a half fold.