Surfactants are one of the most frequently used adjuvants in oral pharmaceutical preparations, used primarily as solubilizers, stabilizers, emulsifiers, and wetting agents. However, surfactants can disrupt normal membrane structure. In this study, lactate dehydrogenase (LDH) and mucus were evaluated as potential markers of intestinal damage in a single-pass in situ perfusion model in the rat. The release of LDH and mucus into the intestinal lumen of the rat following perfusion of the nonionic surfactants Tween 80 and Triton X-100 was determined. The release rate of LDH increased in the order saline < Tween 80 < Triton X-100 in both jejunum and colon. LDH release rate was approximately three times lower in the colon than in the jejunum, but relative effects of nonionic surfactants were comparable between regions. In addition, the rate of LDH release in the jejunum increased with decreasing perfusion rates for both saline and Tween groups and with increasing Tween 80 concentrations. At each flow rate studied, mucus release rate was greater in the presence of Tween 80 and Triton X-100 than saline, but there was no significant difference between the effect of Tween 80 and Triton X-100 on mucus release rate. When perfusion of Triton X-100 was followed by saline, rates of both mucus and LDH release returned to baseline values, suggesting damage is reversible. Histological damage agreed with trends observed in LDH and mucus release rates. This model allows for early evaluation of intestinal damage due to both excipients and active ingredients and simultaneous measurement of drug absorption.