Inflammation is part of the body’s natural defense against harmful stimuli and is a hallmark of many diseases. Characterized by tissue damage, fibrosis, metaplasia and infiltration of immune cells, prolonged inflammation is usually very painful and is often an early warning sign of cancer, particularly in the gut. Proteases have been broadly implicated in inflammation; however, depending on the context, their roles may be pro- or anti-inflammatory. Legumain is a lysosomal cysteine protease that has been associated with a number of inflammatory diseases, including stroke, atherosclerosis, and cancer. The current study investigates the role of legumain in inflammatory bowel disease using the fluorescent activity-based probe, LE28. We have utilized a mouse model of ulcerative colitis induced by dextran sodium sulfate. Using ex vivo fluorescence imaging, we demonstrated that legumain activity is upregulated in the proximal colon of DSS-treated mice compared to healthy controls. This was confirmed by biochemical assessment of LE28 labeling using fluorescent SDS-PAGE and subsequent western blotting for legumain expression. While the normal colon expresses basal levels of active legumain, an additional, higher molecular weight form appears in the inflamed colon. Legumain activity is predominantly found in CD68+ macrophages within the mucosal layers of the colon, with very little activity in the surrounding muscle tissue. Furthermore, low levels of active legumain are secreted into the intestinal lumen and feces of mice with colitis, but not healthy mice. We are currently assessing legumain activity in other mouse models of intestinal inflammation and in biopsies and fecal samples of patients with ulcerative colitis, Crohn’s Disease, and irritable bowel syndrome to establish its value as a biomarker for disease. We are also using legumain inhibitors to assess the contribution of legumain to the stages of initiation, maintenance, and resolution of intestinal inflammation.