There is an increasing need by the tobacco industry to evaluate the oral/buccal irritation potential of tobacco products both to support product development goals and for sound product stewardship. The use of in vitro human cell and tissue-based test methods to replace in vivo animal models addresses the need for more human-relevant predictive tools, and is consistent with many corporate animal welfare policies. Although monolayer cell-based cytotoxicity assays have been used, three-dimensional tissue constructs provide distinct advantages since tissue exposures and pharmacokinetics more closely resemble the in vivo events. This study was conducted to evaluate the EpiOral™ human oral/buccal tissue model (MatTek Corporation) for determining oral irritation of tobacco products. We applied a dilution series of tobacco extracts onto EpiOral™ tissues for various exposure times (2 to 16 hours) and measured cell viability and the synthesis/release of the inflammatory mediators IL-1α and IL-8, relative to an Artificial Saliva-treated control. We measured tobacco extract concentration-related decreases and exposure-time related decreases in viability for the highest tobacco extract concentrations (relative viability was reduced to 32% for the 100% tobacco extract after 16 hours exposure). We also found that increases in IL-1α release (up to 19-fold) generally correlated with reductions in viability, confirming that the exposures likely induced cytotoxic or cytolytic effects and consequent loss of cell membrane integrity. Exposure time-related increases in IL-8 release were generally observed in tissues treated with the three lower tobacco extract concentrations where relative viabilities were generally similar to negative control levels. However, IL-8 release values were below control levels at the highest tobacco extract concentrations where cytotoxic effects inhibited the cells’ ability to synthesize proteins. These IL-8 expression results demonstrate that secondary inflammatory cytokine synthesis can be induced in oral epithelium tissue constructs, particularly at exposures that would not be expected to cause overt cytotoxic effects.