Alterations in intestinal permeability can lead to increased uptake of luminal antigens, which has been linked to several intestinal diseases, such as inflammatory bowel diseases, celiac disease, and irritable bowel syndrome, but also to extra-intestinal diseases. Promising therapies that target intestinal permeability could be developed, for instance tight junction modulators. Consequently, permeability assays are increasingly being used as treatment endpoints in clinical studies. Therefore, reliable, reproducible, and feasible methods for measuring intestinal permeability in the clinical setting are necessary. Currently, a variety of in vivo, ex vivo, and in vitro tests are available, some of which are only applicable to basic research. Despite the various options available to measure gut permeability, their use in clinical setting is still limited because of their heterogeneity. Here, we describe a clinical method to measure intestinal permeability using two non-metabolizable sugars.