Multicellular organisms need epithelial barriers to remain compartmentalized and protected from external influences. Although much progress has been made in understanding barrier integrity disruption in Celiac disease (CD), the regulatory and genetic mechanisms underlying the increased intestinal epithelial flux are still unknown. As we learn more about the regulation of permeability in homeostasis and pathogenesis, we will be able to develop strategies to strengthen the epithelial barrier function in intestinal disorders, including CD. For this purpose, Ussing chambers are increasingly used in native tissue, such as gut mucosa or cell monolayers, to assess the integrity of the barrier. In particular, the Ussing chambers allow the measurement of paracellular and transcellular parameters of CD small intestinal biopsies under physiologically specific conditions. In diverse types of diseases, this method is commonly used to determine epithelial barrier defects, but its application to CD has not yet been widely expanded. To provide a great model of barrier ex vivo studies in CD, we facilitate a standard protocol to measure paracellular and transcellular permeability using the Ussing chamber.