BACKGROUND: The aberrant expression of α defensin 5 (DEFA5) protein in colonic inflammatory bowel diseases (IBDs) underlies the distinct pathogenesis of Crohn's colitis (CC). It can serve as a biomarker for differentiating CC from Ulcerative colitis (UC), particularly in Indeterminate colitis (IC) cases into UC and CC. We evaluated the specificity of commercially available anti-DEFA5 antibodies, emphasizing the need to further validate their appropriateness for a given application and highlighting the necessity for novel antibodies. METHODS: We established two mice monoclonal DEFA5 antibody clones, 1A8 and 4F5, by immunizing mice with purified recombinant protein. We validated the specificity, sensitivity, and cross-reactivity of these antibodies in recognizing both endogenous and recombinant DEFA5 protein, especially for use in Immunohistochemistry (IHC), Western blot (WB), Immunoprecipitation (IP), and enzyme-linked immunosorbent assay (ELISA). RESULTS: Clones 1A8 and 4F5 effectively recognized the endogenous DEFA5 in active human colon tissue from patients with diverticulitis (DV), UC, CC, and IC disease samples, as well as in transiently transfected HEK293T cells expressing DEFA5 with minimal non-confounding cross reactivity. CONCLUSIONS: The 1A8 and 4F5 clones are useful for a wide variety of immunoassays, including WB, IHC, IP/WB, and ELISA. Their specificity enhances their potential as valuable tools for research applications in IBD colitis.