Exposure to cigarette smoke (CS) increases the risk of developing lung diseases including chronic obstructive pulmonary disease (COPD) and cancer. CS is a complex aerosol with over 5000 chemicals, thus it is difficult to determine individual contributions to overall toxicity, as well as the molecular mechanisms by which smoke constituents exert their effects. We selected 14 well-known smoke constituents and established a High Content Screening method in normal human bronchial epithelial cells (NHBE). The impact of the constituents was investigated using 13 multi-parametric indicators of cellular toxicity and complemented with microarray-based transcriptome analysis followed by a computational approach leveraging mechanistic network models to identify and quantify perturbed molecular pathways. Smoke constituents were evaluated in a wide range of concentrations and at different time points. Most constituents induced cellular death only at high doses. Most important toxicity mechanisms were: DNA damage/growth arrest, oxidative stress, mitochondrial stress and apoptosis/necrosis. In summary, combination of standard toxicological endpoints with a systems-based impact assessment allows for a more robust scientific basis for toxicological assessment of smoke constituents, while gaining insight on the molecular mechanisms activated upon exposure this allowed us to establish an in vitro systems toxicology platform which will now be applied to a broader selection of smoke constituents and their mixtures and can serve as the basis for testing the impact of other airborne toxicants on NHBEs.