Using cigarette smoke condensate or phosphate buffered saline (PBS) bubbling are two of the main methods available to evaluate the effects of cigarettes pertaining to in vitro biological studies. However, there are some limitations with such methods. For example, materials in the vapor phase could not be assessed with the cigarette smoke condensate method and it was difficult to evaluate insoluble components with the PBS bubbling method. To solve these potential problems, we assembled an exposure system that can evaluate the biological activity of whole cigarette smoke. The exposure system design is based on the CULTEX™ cultivation technique, which exposes cells to 5 different concentrations of smoke from a specific cigarette simultaneously. A clear dose response relationship between cell viability and total exposure was observed. Furthermore, the system was able to observe the effects of both the particulate phase and the vapor phase. An application study of the whole smoke exposure system using cigarettes with different specifications was performed. In this study, the cytotoxic effects of some types of cigarettes, using the whole smoke exposure system, were evaluated by neutral red uptake and WST-1 assays using an A549 human lung epithelial cell line, and compared by median inhibitory concentration 50 (IC₅₀). As a result, the system was able to detect cytotoxic differences between different cigarette specifications. Therefore, we confirmed the utilization of the whole smoke exposure system as a method to assess one of the biological activities in mainstream cigarette smoke.