Although the list of proteins modified by ubiquitination has been growing rapidly, a reliable method to biochemically isolate and identify in vivo ubiquitinated substrates is needed. Here we describe a proteomic approach to enrich, identify, and validate ubiquitinated proteins from Saccharomyces cerevisiae on a large scale. To facilitate the purification of ubiquitinated proteins, all four ubiquitin genes were knocked out, and a plasmid coding N-terminal His-tagged ubiquitin was introduced in a yeast strain. Ubiquitinated proteins from the strain were purified by metal chelation chromatography under denaturing condition to minimize co-isolation of interacting proteins. Purified proteins were further analyzed by highly sensitive mass spectrometry to determine their identities. The ubiquitination sites in the proteins could be determined in many cases according to the mass shift caused by the modification. Moreover, the polyubiquitin chain topology could be detected by the same method as well. To confirm the genuineness of the identified ubiquitin conjugates, several strategies were developed: (1) to subtract proteins detected in the control experiment (metal chelation purification from wild-type yeast strain); (2) to remove proteins that did not show the increase of apparent molecular weight because of ubiquitination; (3) to accept proteins with identified ubiquitination sites; and (4) to confirm the state of ubiquitination of individual protein by immunoprecipitation and Western blotting analysis. The method provides a generic approach for biochemical characterization of ubiquitinated proteins and can be extended to analyze targets of ubiquitin-like molecules.