Abstract Background and Aims Endoscopy and the use of faecal calprotectin faecal CP are among the least-favoured methods for assessing disease activity by inflammatory bowel disease IBD patients; the handling/processing of faecal samples is also impractical. Therefore, we sought to develop a novel neo-epitope serum calprotectin enzyme-linked immunosorbent assay ELISA, CPa9-HNE, with the aim of quantifying neutrophil activity and neutrophil extracellular trap NET-osis and proposing a non-invasive method for monitoring disease activity in IBD patients. Methods In vitro cleavage was performed by mixing calprotectin S100A9/S100A8 with human neutrophil elastase HNE, and a novel HNE-derived calprotectin neo-epitope CPa9-HNE was identified by mass spectrometry for ELISA development. The CPa9-HNE ELISA was quantified in supernatants from ex vivo activated neutrophils and serum samples from patients with ulcerative colitis UC, n = 43, Crohn’s disease CD, n = 93, and healthy subjects HS, n = 23. For comparison, faecal CP and MRP8/14 biomarkers were also measured. Results CPa9-HNE was specific for activated neutrophils ex vivo. Serum CPa9-HNE levels were 4-fold higher in CD p <0.0001 and UC p <0.0001 patients than in HS. CPa9-HNE correlated well with the Simple Endoscopic Score SES-CD score r = 0.61, p <0.0001, MES r = 0.46, p = 0.0141, and the full Mayo score r = 0.52, p = 0.0013. CPa9-HNE was able to differentiate between CD and UC patients in endoscopic remission and moderate/severe disease activity (CD: area under the curve AUC = 0.82 p = 0.0003, UC: AUC = 0.87 p = 0.0004). The performance of CPa9-HNE was equipotent or slightly better than that of faecal CP. Conclusions Serum CPa9-HNE levels were highly associated with CD and UC patients. CPa9-HNE correlated with the SES-CD score and the full Mayo score, indicating a strong association with disease activity.