One of the goals of the “3Rs principle” is to replace in vivo testing methodologies with in vitro procedures on human cells, followed by computational systems biology modelling to determine toxicological risk. System toxicology, which integrates standard toxicology together with quantitative analysis of large networks of molecular and functional changes, have demonstrated the potential to help realize the 3Rs vision. Omics and high content screening methods represent fundamental pillars of a system toxicology-based approach and at Philip Morris International R&D we have developed a large battery of HCS assays to investigate how specific substances alter the phenotype of a cell grown in submerged conditions. As more physiological cell culture model, such as organotypic 3D culture are also part of our portfolio, we invested in the development of imaging-based approaches which would enable high-content investigations of 3D bronchial tissues. In this seminar, recently developed approach for phenotypic analysis of organotypic 3D Bronchial Tissues will be presented.