The main objective of this review is to discuss recent advancements in the overall investigation and in vivo prediction of drug absorption. The intestinal permeability of an orally administered drug (given the value P-eff) has been widely used to determine the rate and extent of the drug's intestinal absorption (F-abs) in humans. Preclinical gastrointestinal (GI) absorption models are currently in demand for the pharmaceutical development of novel dosage forms and new drug products. However, there is a strong need to improve our understanding of the interplay between pharmaceutical, biopharmaceutical, biochemical, and physiological factors when predicting F-abs and bioavailability. Currently, our knowledge of GI secretion, GI motility, and regional intestinal permeability, in both healthy subjects and patients with GI diseases, is limited by the relative inaccessibility of some intestinal segments of the human GI tract. In particular, our understanding of the complex and highly dynamic physiology of the region from the mid-jejunum to the sigmoid colon could be significantly improved. One approach to the assessment of intestinal permeability is to use animal models that allow these intestinal regions to be investigated in detail and then to compare the results with those from simple human permeability models such as cell cultures. Investigation of intestinal drug permeation processes is a crucial biopharmaceutical step in the development of oral pharmaceutical products. The determination of the intestinal P-eff for a specific drug is dependent on the technique, model, and conditions applied, and is influenced by multiple interactions between the drug molecule and the biological membranes.