In 2007, the National Research Council published a new strategic plan for toxicology assessment to update and advance our knowledge on the toxicity and the mode of action of environmental agents. This 21st Century Toxicology paradigm recommends the use of computational toxicology and systems biology in combination with human-relevant in vitro models that could allow multiple dose testing, reduce the use of animals in research and generate better data on the potential risks to humans. Exposure to cigarette smoke (CS) is known to cause serious diseases including chronic obstructive pulmonary disease. Philip Morris International is working to develop products which could potentially reduce disease risk in smokers who use those products instead of combustible cigarettes. We have applied 21st Century Toxicology approaches among our assessment methods.and started to investigate if human airway in vitro models exposed to CS at the air-liquid interface could mimic the in vivo biological response. Various endpoints (e.g. cytotoxicity, CYP1A1/1B1 enzyme activity, inflammatory markers release, mucus ciliary beating frequency, morphological and transcriptomic changes) were collected at different times after exposure to identify and compare the dose- and time-dependent effect of each exposure conditions. By using systems toxicology-based risk assessment approaches combining computable biological network models and gene expression changes, the molecular perturbations triggered after the different exposure conditions were analyzed and quantified. Using this example of 21st Century Toxicology Assessment, we will discuss the advantages, the limitations and the challenges of such strategies, especially the repeated exposure study designs required to assess respiratory disease in vitro.