Background: Only a few exposure systems are presently available that enable cigarette smoke exposure of living cells at the air–liquid interface, of which one of the most versatile is the Vitrocell® system (Vitrocell® Systems GmbH). To assess its performance and optimize the exposure conditions, we characterized a Vitrocell® 24/48 system connected to a 30-port carousel smoking machine. The Vitrocell® 24/48 system allows for simultaneous exposure of 48 cell culture inserts using dilution airflow rates of 0–3.0 L/min and exposes six inserts per dilution. These flow rates represent cigarette smoke concentrations of 7–100%. Results: By characterizing the exposure inside the Vitrocell® 24/48, we verified that (I) the cigarette smoke aerosol distribution is uniform across all inserts, (II) the utility of Vitrocell® crystal quartz microbalances for determining the online deposition of particle mass on the inserts, and (III) the amount of particles deposited per surface area and the amounts of trapped carbonyls and nicotine were concentration dependent. At a fixed dilution airflow of 0.5 L/min, the results showed a coefficient of variation of 12.2% between inserts of the Vitrocell® 24/48 module, excluding variations caused by different runs. Although nicotine and carbonyl concentrations were linear over the tested dilution range, particle mass deposition increased nonlinearly. The observed effect on cell viability was well-correlated with increasing concentration of cigarette smoke. Conclusions: Overall, the obtained results highlight the suitability of the Vitrocell® 24/48 system to assess the effect of cigarette smoke on cells under air–liquid interface exposure conditions, which is closely related to the conditions occurring in human airways