The intestinal mucosa is responsible for the absorption of nutrients from the lumen and for the separation of the potentially toxic luminal content (external environment) from the host (internal environment). Disruption of this delicate balance at the mucosal interface is the basis for numerous (intestinal) diseases. Experimental animal studies have shown that gut wall integrity loss is involved in the development of various inflammatory syndromes, including post-operative or post-traumatic systemic inflammatory response syndrome, sepsis, and multiple organ failure. Assessment of gut wall integrity in clinical practice is still a challenge, as it is difficult to evaluate the condition of the gut non-invasively with currently available diagnostic tools. Moreover, non-invasive, rapid diagnostic means to assess intestinal condition are needed to evaluate the effects of treatment of intestinal disorders. This review provides a survey of non-invasive tests and newly identified markers that can be used to assess gut wall integrity.