Shotgun proteomics is a technique now being used frequently to analyze protein mixtures because of its ability to directly analyze a complex protein sample through a multidimensional liquid chromatography approach that utilizes differences in charge and hydrophobicity. In this study, we used this technique to identify proteins that are ubiquitinated in Saccharomyces cerevisiae. The 76 amino acid ubiquitin protein has been shown to regulate many cellular processes by attaching post-translationally to other proteins. In order to gain a full understanding of ubiquitin biology, we have begun to comprehensively catalogue ubiquitin substrates in S.cerevisiae. To do this, we grew yeast strains expressing His6-tagged ubiquitin, and utilized the His6 tag to affinity purify ubiquitin-protein conjugates from yeast protein lysates. The affinity purification was proteolytically digested with trypsin, fractionated using online multidimensional chromatography, and then analyzed by tandem mass spectrometry. Functional classification of the identified 616 ubiquitinated substrates according to gene ontology (GO) annotations indicates that they are involved in a variety of cellular processes, including many metabolic pathways. Modification of proteins by ubiquitin has been linked to the pathogenesis of many diseases including cancer and Alzheimer's; knowledge of all of the targets of ubiquitination generated by studies such as this may eventually provide avenues for therapeutic intervention.