Decellularization of the human intestine in patients with inflammatory bowel disease (IBD) provides an experimental platform for the investigation of the bowel extracellular matrix (ECM). Here, we present a protocol for removing cellular components from the IBD intestinal tissue while preserving its structure and ECM components. We describe steps for pre-processing specimens and decellularization by osmotic pressure difference, followed by detergents and enzymes. We then detail procedures for extensive washing. The decellularized tissue can be stored or processed according to the experimental requirements. For complete details on the use and execution of this protocol, please refer to Lin et al.(1).