Gastrointestinal toxicity is a major concern in the development of drugs. Here, we established a scalable organotypic platform to screen drugs for toxicity in murine small and large intestinal tissues. As a proof-of-concept, we applied this platform to assess gastrointestinal toxicity of ~50 clinically used oncology drugs, encompassing diverse mechanisms of action. Nearly all tested drugs had a deleterious effect on the gut, with increased sensitivity in the small intestine. The identification of differential toxicity between the small and large intestine enabled us to pinpoint differences in expression of drug uptake transporters (antifolates), drug-metabolizing enzymes (cyclophosphamide) and drug target expression (EGFR inhibitors) across the gut. These results highlight an under-appreciated distinction between small and large intestine toxicity and suggest distinct tissue properties important for modulating drug-induced gastrointestinal toxicity. Moreover, our platform enables spatial and mechanistic prediction for how drugs affect the gut, accelerating the development of safer therapeutics.