BACKGROUND: The main challenge in new drug development is accurately predicting the human response in preclinical models. METHODS: In this study, we developed three different intestinal barrier models using advanced biofabrication techniques: (i) a manual model containing Caco-2 and HT-29 cells on a collagen bed, (ii) a manual model with a Caco-2/HT-29 layer on a HDFn-laden collagen layer, and (iii) a 3D bioprinted model incorporating both cellular layers. Each model was rigorously tested for its ability to simulate a functional intestinal membrane. RESULTS: All models successfully replicated the structural and functional aspects of the intestinal barrier. The 3D bioprinted intestinal model, however, demonstrated superior epithelial barrier integrity enhanced tight junction formation, microvilli development, and increased mucus production. When subjected to Ibuprofen, the 3D bioprinted model provided a more predictive response, underscoring its potential as a reliable in vitro tool for drug toxicity testing. CONCLUSION: Our 3D bioprinted intestinal model presents a robust and predictive platform for drug toxicity assessments, significantly reducing the need for animal testing. This model not only aligns with ethical testing protocols but also offers enhanced accuracy in predicting human responses, thereby advancing the field of drug development.