Over the past three decades, the genotoxic effects of cigarette smoke have generally been evaluated in non-human cell models after exposure to particulate phase, gas phase, or cigarette smoke condensate, rather than the whole smoke aerosol itself. In vitro setups using human cell lines and whole smoke exposure to mimic actual aerosol exposure should more accurately reflect human cigarette smoke exposure. We investigated the VITROCELL® 24 air–liquid interface exposure system in combination with the comet assay to assess DNA damage in two different human lung epithelial cell lines exposed to whole smoke. Results showed a repeatable and reproducible dose–response relationship between DNA damage and increased whole smoke dose in both cell lines. Thus, the combination of the comet assay with the VITROCELL® 24 represents a valuable new in vitro test system to screen and assess DNA damage in human lung cells exposed to whole smoke.