Postoperative ileus (POI) is a frequent and severe complication after intestinal surgery (1, 2). Gastrointestinal dysfunction originates from an inflammatory response within the tunica muscularis induced by the surgical handling of the intestine (3). Resident macrophages have been shown to play a key role in the initiation of POI (4, 5). Furthermore, this inflammation can spread from manipulated bowel segments to unmanipulated distant parts of the gastrointestinal (GI) tract (6). Herein we describe the functional assessment of in vivo intestinal motility in a standardized mouse model of POI. This method provides information about the strength of POI and the spreading of the underlying inflammation along the GI tract. Initially, animals are laparotomized and small bowel becomes intestinally manipulated (15 min). Ninety minutes before experiment´s end point, colonic transit is analyzed (5-10 min) by the excretion time of a transanally inserted probe. Subsequently, GI transit is measured by means of distribution of an orally administered fluorescent dye along the GI tract over 90 minutes. GI transit analysis requires 30 minutes. Combining both methods in one animal offers the advantage to analyze motility of the complete GI tract, while former studies often focused solely on separated bowel motility or gastric emptying.