In the last few years tobacco companies have been developing several research strategies in order to reduce the risks associated with smoking. These strategies include, for example, the refining of alternative cigarette designs that reduce the amount of hazardous chemicals in the mainstream smoke by introducing modified filters, and/or reducing the amount of biologically significant ingredients in tobacco-burning cigarettes. In the last few decades numerous studies have been published to assess the biological activity of tobacco smoke using in vivo and in vitro test systems. In this scenario a general scientific consensus on how to measure and characterize the risk associated with cigarette smoke is still lacking. Short-term in vitro assays, which are widely accepted by regulatory agencies around the world, are useful tools to evaluate both the biological activity and the progress towards a reduction of tobacco smoke toxicity. These assays could be mainly applied to evaluate cytotoxicity and genotoxicity properties on whole cigarette smoke as well as condensates or fractions of whole smoke. Cytotoxicity induction can be measured as cellular viability and growth rates using different end-points. Otherwise, the target of genotoxicity studies is the DNA molecule. For genotoxicity evaluation, the end-points and cell systems should be chosen from those that are relevant and appropriate as clinical surrogate markers. In this respect, the occurrence of early biological effect markers, such as mutational or clastogenic events (point mutations, frameshifts, micronuclei, SCE, DNA adducts) in bacterial and mammalian cells should be studied in a tiered approach following the guidelines of regulatory agencies. The choice of criteria shall be matter of discussion.