Fission and budding yeast have been regarded as valuable tools for studying several cellular processes in eukaryotic cells and have been exploited as model systems for the identification of determinants of chemosensitivity. Indeed, yeast mutants of DNA repair and cell cycle checkpoint pathways exhibit increased sensitivity to selected antitumor drugs, thereby allowing us to establish the role of specific genes in drug response. The basic cellular functions of simple eukaryotic organisms and mammalian cells are conserved. Thus, the features of yeast, such as a small genome, a fast growth rate, and a peculiar life cycle, which allows easy genetic manipulation, can provide advantages in the identification of determinants of chemosensitivity. Here we focus on methods developed in fission yeast with particular reference to gene disruption, transformation, and mutagenesis approaches. These methods could be useful in an attempt to develop target-specific therapeutic strategies.