Preclinical drug absorption studies are a key step when developing new therapeutics as they determine how much of an oral drug reaches the systematic circulation. Yet, current industry-standard in vitro gut models, used to predict drug permeability across the human intestinal barrier, often fail to recapitulate the physiology of the human gut; making them poor predictors of the gut permeability. In recent years, efforts have been put into improving those in vitro models, in particular with the use of microphysiological system (MPS), also known as Organ-on-a-Chip (OOC). MPS aim to better represent the physiology, structure and functions of human tissues and organs. In this Application Note, we show how we developed and characterised a gut MPS closely aligned to the human small intestine, using the PhysioMimix™ Single-Organ system, and demonstrated its utility to predict drug permeability.