The yeast Saccharomyces cerevisiae became the first eukaryotic organism to have its entire genome sequenced (1). With the completion of the genome, over 6000 genes on 16 chromosomes were identified. Several laboratories are undertaking the task of identifying the yeast proteins on two-dimensional (2-D) gels with the goal of developing a yeast protein database for studying global changes in protein synthesis, protein modification, and protein degradation that result from environmental or genetic changes (2,3). This chapter provides protocols for labeling yeast with ³⁵S-methionine and the preparation of 2-D extract using methods developed by Garrels et al. and Boucherie et al. (2,3). Additional protocols for preparing yeast 2-D extracts can be found at the Geneva University Hospital’s Electrophoresis Laboratory, which can be accessed via the World Wide Web at http://expasy.hcuge.ch/ch2d/technical-info.html and at the University of Göteberg’s Lundberg Laboratory, which can be accessed at http://yeast-2dpage.gmm.gu.se/sacch/immobiline/methods/labelling.