Significant studies have been carried out to understand effective management of intestinal fibrosis. However, the lack of better knowledge of fibrosis has hindered the development of a preventative drug. Primarily, finding a suitable animal model is challenging in understanding the mechanism of Crohn's-associated intestinal fibrosis pathology. Here, we adopted an effective method where TNBS chemical exposure to mice rectums produces substantially deep ulceration and chronic inflammation, and the mice then chronically develop intestinal fibrosis. Also, we describe a technique where a rapamycin injection shows inhibitory effects on TNBS-mediated fibrosis in the mouse model. To assess the underlying mechanism of fibrosis, we methodically discuss a procedure for purifying Cx3Cr1+ cells from the lamina propria of TNBS-treated and control mice. This detailed protocol will be helpful to researchers who are investigating the mechanism of fibrosis and pave the path to find a better therapeutic invention for Crohn's-associated intestinal fibrosis.