In vitro toxicology studies of tobacco and tobacco smoke have been used to understand why tobacco use causes cancer and to assess the toxicologic impact of tobacco product design changes. The need for toxicology studies has been heightened given the Food and Drug Administration's newly granted authority over tobacco products for mandating tobacco product performance standards and evaluate manufacturers' health claims about modified tobacco products. The goal of this review is to critically evaluate in vitro toxicology methods related to cancer for assessing tobacco products and to identify related research gaps. PubMed database searches were used to identify tobacco-related in vitro toxicology studies published since 1980. Articles published before 1980 with high relevance also were identified. The data were compiled to examine (a) the goals of the study, (b) the methods for collecting test substances, (c) experimental designs, (d) toxicologic end points, and (e) relevance to cancer risk. A variety of in vitro assays are available to assess tobacco smoke that address different modes of action, mostly using non-human cell models. However, smokeless tobacco products perform poorly in these assays. Although reliable as a screening tool for qualitative assessments, the available in vitro assays have been poorly validated for quantitative comparisons of different tobacco products. Assay batteries have not been developed, although they exist for nontobacco assessments. Extrapolating data from in vitro studies to human risks remains hypothetical. In vitro toxicology methods are useful for screening toxicity, but better methods are needed for today's context of regulation and evaluation of health claims.