Neutrophils play a significant role in sustaining chronic inflammation in Inflammatory Bowel Disease. The intestinal basement membrane acts as a barrier for immunological homeostasis, where the α3 and α4 chains of type IV collagen are expressed on the mucosal surface. We wanted to develop a biomarker reflecting early tissue injury, providing an opportunity for intervention. Two competitive enzyme-linked immunosorbent assays (ELISAs) quantifying human neutrophil elastase (HNE) degraded neo-epitopes of COL4A3 and COL4A4 were developed and investigated in two observational cohorts (n = 161, n = 100). A biomarker of MMP-mediated degradation of COL4A1 (C4M) was used for comparison. In Cohort 1, patients with mild endoscopic ulcerative colitis showed elevated levels of C4A3-HNE compared to those with severe disease. C4M had a strong positive correlation with disease activity. C4A3-HNE/C4M provided superior discrimination between mild and severe endoscopic disease and negatively correlated to disease activity. In Cohort 2, C4A4-HNE and C4A4-HNE/C4M showed similar trends. C4A3-HNE and C4A4-HNE possibly reflect early intestinal tissue injury. Combining the markers with a biomarker of another α-chain of the same collagen provides information on two distinct stages of mucosal damage. These biomarkers may be used to monitor disease flare-up in patients in remission, reducing the need for frequent endoscopic procedures.