Background Classical two‐dimensional (2D) cell culture as a drug or nanoparticle test system only poorly recapitulates in vivo conditions. Animal studies are costly, ethically controversial, and preclude large‐scale testing. Methods and Results We established a three‐dimensional (3D) tissue slice air–liquid interface (ALI) culture model for nanoparticle testing. We developed an optimized procedure for the reproducible generation of large sets of tissue slices from tumor xenografts that retain their tissue architecture. When used for the analysis of nanoparticles based on chemically modified polyethylenimines (PEIs) to deliver siRNA or DNA, differences in transfection efficacy and cytotoxicity between nanoparticles were observed more clearly than in 2D cell culture. While nanoparticle efficacies between cell culture and the tissue slice model overall correlated, the tissue slice model also identified particularly suitable candidates whose efficacy was underestimated in 2D cell culture and had already been shown in previous in vivo studies. Conclusion The ex vivo 3D tissue slice ALI culture model is a powerful system that allows the effective evaluation of biological nanoparticle efficacy and biocompatibility in an intact tissue environment. It is comparably inexpensive, time‐saving, and follows the 3R principle, while allowing the identification of critical nanoparticle properties and optimal candidates for in vivo applications.