There has been rapid progress in approaches to assess the effects of cigarette whole smoke in vitro, enabling the exposure of cells at the air-liquid-interface (ALI) rather than in submerged conditions. Only a few exposure systems are available to date allowing ALI exposure of cells in culture and one of the most advanced is the VITROCELL® system (VITROCELL Systems GmbH). It includes a technique to assess the applied dose of whole smoke in situ. To better understand its performance and to optimize the experimental conditions when exposing cells at the ALI we recently characterized a VITROCELL® system connected to 30-port carousel smoking machine. The system (VITROCELL 24/48) allows for simultaneous exposure of 48 cell culture inserts with undiluted smoke or up to a maximum of 8 different smoke dilutions, with humidified air using flow rates of 0.1 – 3.0 l/min and exposing 6 inserts per dilution (n=6). These flow rates translate into smoke dilution ratios of 1:1.57 – 1:18.14 (smoke:air v/v) or 63-5.6% of whole smoke respectively. The study results demonstrated equal aerosol distribution across all 48 inserts and the utility of VITROCELL crystal quartz microbalances (QCM) for determining the online deposition of smoke particles on the inserts. The resolution of the QCM is 10 nanogram/cm2s, providing high sensitivity. The well-to-well variability determined in the VITROCELL module after whole smoke exposure was +/-10%, at a fixed smoke dilution flow of 0.5 l/min. Using different dilution flow rates of 0.1 – 3.0 l/min, a dose-dependent smoke exposure of the inserts could be demonstrated, determined by particle deposition on the QCM. In addition, we applied a colorimetric readout to confirm the dose-dependent exposure, which was achieved using an oxidation sensitive reagent (tetrazolium) added to empty cell culture inserts. Finally, cellular cytotoxicity of undiluted whole smoke as well as diluted whole smoke (dilution flow rates between 0.1 – 1.0 l/min) was determined by a resazurin assay using lung epithelial BEAS-2B cells, grown in the inserts. Again, a dose-dependent and reproducible decrease in cellular viability was identified, highlighting the suitability of the VITROCELL system to assess the effect of cigarette smoke on cells under exposure conditions more closely related to those occurring in human airways.