Distillers dried grains with solubles (DDGS) are co-product of dry-grind ethanol plant, which can be used as a feedstock for fermentation, e.g. for biofuels or other value-added products. However, DDGS loading is a critical factor in the pretreatment, hydrolysis, and fermentation, where the low DDGS loading lowers sugar concentration in the hydrolysate and yields low product concentrations, which can result in high energy cost at the recovery step, while too high loading may have inhibitory effects on the microbial growth during fermentation. Therefore, an optimal DDGS loading needs to be determined at the pretreatment step for high sugar yields. In this study, dilute sulfuric acid pretreatment of DDGS was optimized to obtain the high release of sugars with the use of high DDGS loading at an appropriate acid concentration. An experimental design was constructed with the Box-Behnken response surface method using the ranges of 5-20% (wt) DDGS, 1-5% (wt) H2SO4, and 20-60 min pretreatment times at a constant temperature of 120 degrees C. The results revealed the highest yields of sugars (0.39 g/g) at 20% DDGS, 5% (wt) H2SO4, and 120 degrees C after 1 h. The results justify complete hydrolysis of hemicellulose and residual starch fractions and provide sufficient amount of sugars for the fermentation process.