The study of intestinal permeability is gaining growing interest due to its relevance in the onset and progression of several gastrointestinal and non-gastrointestinal diseases. Though the involvement of impaired intestinal permeability in the pathophysiology of such diseases is recognized, there is currently a need to identify non-invasive biomarkers or tools that are able to accurately detect alterations in intestinal barrier integrity. On the one hand, promising results have been reported for novel in vivo methods based on paracellular probes, i.e., methods that can directly assess paracellular permeability and, on the other hand, on fecal and circulating biomarkers able to indirectly assess epithelial barrier integrity and functionality. In this review, we aimed to summarize the current knowledge on the intestinal barrier and epithelial transport pathways and to provide an overview of the methods already available or currently under investigation for the measurement of intestinal permeability.