Mucositis is a debilitating side-effect of chemotherapy which affects the mucosa of the gastrointestinal tract, particularly the small intestine. Currently there are no simple, noninvasive methods to detect and monitor small intestinal function and the severity of mucosal damage. Activity of the brush-border enzyme sucrase provides an indicator of small intestinal absorptive function that remains relatively constant throughout life. Measuring 13CO2 levels in expired breath following ingestion of 13C-sucrose is a non-invasive marker of total intestinal sucrase activity. We evaluated the sucrose breath test (SBT) as an indicator of small intestinal injury and dysfunction, utilizing a rat model of chemotherapy-induced mucositis. SBT results reflected the time-course of damage and repair after methotrexate (MTX) treatment, with damage most severe 72 h after chemotherapy, and repair commencing after 96 h. SBT results correlated significantly with jejunal sucrase activity determined biochemically (r2= 0.89; p < 0.005). Moreover, calcium folinate ingested prior to chemotherapy totally prevented damage to the small intestinal mucosa induced by MTX, as assessed by the SBT in concert with structural, and biochemical indices. The SBT provides a simple, non-invasive, integrated measure of small intestinal damage and function. The SBT holds significant potential to monitor small intestinal function in cancer patients undergoing chemotherapy. This technique possesses further applicability to the screening of newly-developed agents for potential gastrointestinal toxicity including the development of new therapies targeted at minimising or preventing the onset of chemotherapy-induced mucositis.