During germination, the aleurone layer of cereal grains is stimulated by gibberellic acid (GA) to secrete most of the enzymes required to degrade the carbohydrate and protein storage products of the endosperm. The complement of enzymes secreted by the aleurone layer is complex, and many specific isoforms have yet to be characterized in barley, wheat and other important cereals. Thus new approaches to study germination-related enzyme activities are required. Activity-based protein profiling (ABPP) utilizes chemical probes that bind specifically to enzymes only in their active form. An activity profile of many enzymes in a single sample can be obtained by the use of probes with distinct specificities examined under different conditions. We used different ABPP probes to determine the activity of a range of enzymes extracted from aleurone layers isolated from grains of a commercial malting barley variety incubated with or without gibberellic acid (GA). Using ABPP, enzymes found to be induced by GA were aleurain-like proteases, cathepsin-B-like proteases and a range of serine hydrolases. By using a panel of monosaccharides as potential product inhibitors, a specific active retaining β-glycosidase in the barley aleurone was identified as a putative xylanase. Our results show that ABPP can be used rapidly to identify a variety of active enzyme isoforms in cereal aleurone without the need for enzyme purification.