As environmental concerns increase in the U.S., there is a push to go “green”. This has fueled the development of ethanol production in the U.S. as a renewable source of energy. In the U.S., corn is used to produce ethanol. Therefore, displacement of corn used in the animal industry to ethanol production has, inevitably caused an increase in the price of corn. Replacing conventional ingredients, such as corn, in monogastric diets with lower cost by products can be attractive economically. The objective of this study was to determine if exogenous enzymes that free reducing sugars from fiber will increase the dispensability of bound nutrients in the economically favorable by-product of ethanol production, distillers dried grains with solubles (DDGS). Effects of non-starch polysaccharidase (NSPase) inclusion rates and combinations with phytase on the ability to release reducing sugars were investigated. Distillers dried grains with solubles was tested in vitro in combination with hemicellulase (0, 0.1, 1, and 10 U/g of DDGS), cellulase (0, 1.5, 15, and 150 U/g of DDGS), xylanase (0, 2.5, 25, and 250 U/g of DDGS), glucanase (0, 0.006, 0.06, and 0.6 U/g of DDGS), and phytase (0, 250, 500, and 1,000 FTU/kg of DDGS). Results indicated an inhibitory effect of phytase on the ability of NSPases to release reducing sugars from DDGS (P = 0.0004). A second study was conducted to determine individual enzyme kinetics of NSPase on pure substrates when combined with increasing concentrations of phytase. Cellulase (P = 0.03), xylanase (P = 0.09), and -glucanase (P = 0.06) combined with increasing concentrations of phytase showed a reduction in the velocity of reducing sugars release from pure substrate. However, we speculate that due to the inability to reach Vmax, the Lineweaver-Burk results were inconclusive. The 1/Km was calculated for all enzymes, and the presence of phytase increased 1/Km for xylanase (P = 0.006). However, -1/Km did not change for cellulase (P = 0.62), -glucanase (P = 0.20), and hemicellulase (P = 0.13). In addition, the 1/Vmax was calculated for all enzymes, and the presence of phytase decreased 1/Vmax for cellulase (P = 0.03) and glucanase (P = 0.01). However, the presence of phytase resulted in no change for the 1/Vmax for xylanase (P = 0.81) and hemicellulase (P = 0.14). The slopes of the regression lines for the Lineweaver-Burk plots showed no effect of the presence of phytase for cellulase (P = 0.40) and hemicellulase (P = 0.27). However, the presence of phytase decreased the linear slope for xylanase (P = 0.006), and increased the linear slope for glucanase (P = 0.006). In summary, phytase appears to act as an inhibitor of NSPase activity in the in vitro digestion on DDGS; however, inconsistent results from the inhibition curves preclude us from determining the type of inhibition.