High drug attrition rates due to toxicity, the controversy of experimental animal usage, and the EU REACH regulation demanding toxicity profiles of a high number of chemicals demonstrate the need for new, in vitro toxicity models with high predictivity and throughput. Metabolism by cytochrome P450s (P450s) is one of the main causes of drug toxicity. As some of these enzymes are highly polymorphic leading to large differences is metabolic capacity, isotype-specific test systems are needed. In this review, we will discuss the use of yeast expressing (mammalian) P450s as a powerful, additional model system in drug safety. We will discuss the various cellular model systems for bioactivation-related toxicity and subsequently describe the properties of yeast as a model system, including the endogenous bioactivation enzymes present, the heterologous expression of (mammalian) P450s and the application of yeasts expressing heterologous P450s and/or other biotransformation enzymes in toxicity studies. All major human drug-metabolizing P450s have been successfully expressed in yeast and various mutagenicity tests have been performed with these humanized yeast strains. The few examples of non-mutagenic toxicity studies with these strains and of the combination of P450s with phase II or other human enzymes show the potential of yeast as a model system in metabolism-related toxicity studies. The wide variety of genome-wide screens available in yeast, combined with its well-annotated genome, also facilitate follow-up studies on the genes involved in toxicity. Unless indicated otherwise "yeast" will refer to baker's yeast Saccharomyces cerevisiae.