Comprehensive analysis of yeast as a model system requires to reliably determine its composition. Systematic approaches to globally determine the abundance of RNAs have existed for more than a decade and measurements of mRNAs are widely used as proxies for detecting changes in protein abundance. In contrast, methodologies to globally quantitate proteins are only recently becoming available. Such experiments are essential as proteins mediate the majority of biological processes and their abundance does not always correlate well with changes in gene expression. Particularly translational and post-translational controls contribute majorly to regulation of protein abundance, for example in heat shock stress response. The development of new sample preparation methods, high-resolution mass spectrometry and novel bioinfomatic tools close this gap and allow the global quantitation of the yeast proteome under different conditions. Here, we provide background information on proteomics by mass-spectrometry and describe the practice of a comprehensive yeast proteome analysis.