This article provides a brief overview of well-established in vivo and in vitro methods that have contributed the most to the understanding of transport processes across the gastrointestinal epithelium. In vivo perfusion techniques in humans revolve around double- and triple-lumen per oral tubes. In animals, in vivo techniques include the single and recirculation perfusion techniques and the double-isotope technique for measurement of net absorption. In vitro methods of studying intestinal transport include the everted gut sac technique, the Ussing chamber, the use of isolated epithelial cells, and the use of brush border and basolateral membranes isolated from enterocytes. The use of fluorescent probes for the measurement of intracellular ionic concentrations is a new and powerful in vivo technique that is now being applied to the gastrointestinal tract.