Acid whey, a byproduct of Greek yogurt, is a significant disposal challenge for the dairy industry. Current acid whey utilization schemes include ethanol production. Since Saccharomyces cerevisiae cannot utilize lactose, the main sugar of acid whey, enzymes or non-traditional yeast strains need to be used. These methods are expensive, and therefore, an alternative approach is needed. A β-galactosidase (β-gal) with activity for lactose has been isolated from barley. Hydrolysis of lactose into glucose and galactose by β-gal would allow for the incorporation of acid whey as a fermentable sugar source in beer production. The objective of this study was to evaluate whether a barley mash at β-gal’s optimum temperature of 40°C, would result in detectable hydrolysis of lactose in acid whey. A mash containing 250 mL of acid whey and 65.9 g of barley meal was shaken constantly at 40°C for 3 h. A control mash consisting of barley meal and water, with no source of lactose added, was used to determine the amount of free glucose in the grain or released from barley amylase activity. Samples were taken at 0 and 180 min and heated to 70°C for 5 min to stop further enzyme activity. Levels of glucose in the samples were analyzed via an enzymatic assay to indicate lactose hydrolysis. Triplicate samples were taken at each time point and the experiment was repeated 3 times. A student’s t-test was conducted to determine significant differences between mean glucose levels in the treatment and the control. At 0 min the control contained 0.03 ± 0.01 g/L glucose and increased to 0.63 ± 0.01 g/L glucose after 180 min. The treatment started at 0.14 ± 0.02 g/L glucose and increased to 4.65 ± 0.17 g/L glucose after 180 min. The level of glucose in the treatment after 180 min was significantly different (P < 0.05) from the control. These results indicate that indigenous enzymes in a barley mash can sufficiently hydrolyze lactose in acid whey. This gives opportunity for utilizing the yogurt byproduct as a raw material in the brewing industry. Further research will look into process development for optimal enzyme activity.