The intestinal barrier is essential in human health and constitutes the interface between the outside and the internal milieu of the body. A functional intestinal barrier allows absorption of nutrients and fluids but simultaneously prevents harmful substances like toxins and bacteria from crossing the intestinal epithelium and reaching the body. An altered intestinal permeability, a sign of a perturbed barrier function, has during the last decade been associated with several chronic conditions, including diseases originating in the gastrointestinal tract but also diseases such as Alzheimer and Parkinson disease. This has led to an intensified interest from researchers with diverse backgrounds to perform functional studies of the intestinal barrier in different conditions. Intestinal permeability is defined as the passage of a solute through a simple membrane and can be measured by recording the passage of permeability markers over the epithelium via the paracellular or the transcellular route. The methodological tools to investigate the gut barrier function are rapidly expanding and new methodological approaches are being developed. Here we outline and discuss, in vivo, in vitro and ex vivo techniques and how these methods can be utilized for thorough investigation of the intestinal barrier.