(1) Background: In the current highly competitive brewing industry, most breweries may benefit from a reduction in mashing time. In this study, a novel enzymatic assay format was used to investigate the activities of α-amylase and β-amylase during different mashing profiles, with the aim to use it as a tool for optimizing the production time of an existing industrial mashing process; (2) Methods: Lab-scale mashings with eight different time-temperature programs and two different pilot brews were analyzed in terms of enzymatic activity, sugar composition, alcohol by volume in the final beer, FAN and others; (3) Results: A 20-min reduction (out of an original 73-min mashing program) was achieved by selecting a temperature profile which maintained a higher enzymatic activity than the original, without affecting the wort sugar composition and fermentability, or the ethanol concentration and foam stability of the final beer. (4) Conclusions: A method is presented which can be used by breweries to optimize their mashing profiles based on monitoring α-amylase and β-amylase activities.