The VITROCELL VC10^® smoke exposure system offers multiple platforms and air liquid interface (ALI) exposure to mimic in vivo-like conditions for assessing the toxicological impact of smoke in in vitro assays. The validation plan to ensure the system and corresponding assays were fit-for-purpose consisted of equipment qualification, pre-validation method development and method validation for neutral red uptake (NRU), sister chromatid exchange (SCE) and Ames assay (TA98, TA100). Parameters assessed for establishing the experimental model consisted of 1) optimization of mammalian and bacterial cell growth at ALI in static and flowing air conditions; 2) investigation of pH changes during exposure; and 3) determination of smoke concentrations and appropriate positive controls for all assay types. In each validation protocol, at least six experiments were performed. Smoke airflows of 10, 8, 6, 4 or 2 L/min and air-control were used for evaluating the assays, and acceptance criteria were established accordingly. NRU acceptance criteria were established to be 1) coefficient of variance is <15% in optical density (540 nm;OD₅₄₀) values between chamber replicates; 2) the positive control elicits >50% decrease in NRU relative to the air control; 3) mean OD₅₄₀ of all air control replicates is >0.2 and 4) viability values above and below the IC₅₀ are required. Acceptance criteria for the Ames assays were set as follows: 1) ALI control counts fall within specified ranges for TA98 and TA100; and 2) positive controls induce ≥2.0 fold increases in revertant numbers over the ALI control. SCE assay acceptance criteria were set as follows: 1) the average number of SCE/cell in the incubator and air controls is within a specified range; 2) at least one positive control treatment exhibits ≥ doubling in the average number of SCE/cell over the incubator and air controls; 3) ≥15 cells must be scored for at least two chamber replicates and 4) data must be generated from at least three non-zero concentrations. Through critical function and assay assessments via the installation, operational, performance and validation protocols, the VC10 was deemed fit-for-purpose and has been shown to induce exposure-related changes in cell survival, sister chromatid exchange and bacterial mutagenesis. Collectively, these criteria support the validity of the use of the VC10 system with several in vitro genetic toxicological assays.