Cigarette smoke (CS) is etiologically linked to fatal respiratory diseases. To enable the development of modified risk tobacco products (MRTPs) to reduce the risk of smoking related disease, there is a need to understand the mechanisms whereby CS causes damage. As an alternative to animal experimentation, the principles of 3 R’s, necessitate the establishment of reliable in vitro models, which may also provide a better predictability of exposure response, when derived from human cells. Among the in vitro models, the three-dimensional organotypic pseudo-stratified culture systems better mimic the morphological, physiological, and molecular attributes of human respiratory tract than primary or immortalized cells cultured as a submerged monolayer. The bronchus and lung parenchyma are the primary sites that manifest smoking related respiratory diseases, however, nasal epithelium has been proposed as a surrogate tissue to study smoking effects on the respiratory tract. We are currently evaluating the utility of organotypic culture systems derived from upper and lower respiratory tract in the assessment of exposure response to CS and prototypic MRTPs. Repeated whole CS exposure showed that both bronchial and nasal organotypic cultures tolerated in a similar manner the two CS concentrations (10% and 16%), measured by tissue integrity (trans epithelial electric resistance) and cellular toxicity (lactate dehydrogenase) assays. By combining the transcriptomic data with computable biological network models, we further showed that several biological processes, including those related to cellular stress response and proliferation, were perturbed in both nasal and bronchial tissues in response to smoke exposure. In summary, the study gives valuable insight into the effect of repeated whole CS exposure on organotypic models derived from both the upper and lower respiratory tract.