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Drug-induced gastrointestinal (GI) toxicity is common, dose-limiting and difficult to predict using conventional Caco-2-based assays that lack physiological relevance. Here we evaluate a transepithelial electrical resistance (TEER) assay using nontransformed human intestinal epithelial cells (hIECs), derived from human pluripotent stem cells, which superiorly recapitulated epithelial diversity and polarity as well as intestinal barrier function. Across 17 clinically relevant compounds (cell cycle inhibitors, tyrosine kinase inhibitors and nonsteroidal anti-inflammatory drugs), the hIEC TEER assay outperformed ATP cell viability assays, and the Caco-2 TEER assay (AUC of 0.96 for hIEC TEER, 0.72 for Caco-2 TEER and ≤0.69 for cell viability assays) correlated with integrated GI toxicity scores using a ≥50% TEER reduction cutoff (sensitivity 92%, specificity 100% and accuracy 94%). Drug exposure was quantified by calculating the margin of safety (IC15:Cmax) and a lumen–surrogate margin of safety for oral agents. For mechanistic insight, transcriptomic analysis using representative chemotherapeutics (paclitaxel and docetaxel) showed the downregulation of cytoskeleton-related pathways, including cytoskeleton in muscle cells, cell adhesion molecules and extracellular matrix–receptor interaction, linking microtubule-targeting chemotherapy to intestinal barrier impairment. This platform provides a robust tool that combines predictive accuracy with the evaluation of cytoskeleton-mediated barrier impairment, enabling the early identification of drug-induced GI toxicity.

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