© ASABE 2021. All rights reserved.Hydrolytic enzymes such as cellulases and xylanases are required for second-generation bioethanol production. Distillers Dried Grains with Solubles (DDGS) has high fiber content which can be used to produce such enzymes by various fungal species. Aspergillus niger is one of the top producers of hydrolytic enzymes. The addition of nitrogen sources can further enhance enzyme production in DDGS based media. Therefore, in this study, two A. niger strains, NRRL 330 and NRRL 567, were analyzed for their potential to enhance hydrolytic enzyme production with the help of nitrogen source optimization. Response Surface Methodology (RSM) was used to optimize peptone, yeast extract and ammonium sulfate for maximum enzyme production. Optimization of different nitrogen sources increased cellulase production from 0.174 to 1.3 IU/ml and hemicellulase production from 9.4 IU/ml to 49.74 IU/ml within 6 days. In conclusion, the optimization of all three nitrogen sources improved both cellulase and hemicellulase production in DDGS-based media.