SUMMARY Ubiquitination is one of the most prevalent protein posttranslational modifications in eukaryotes, and its malfunction is associated with a variety of human diseases. Despite the significance of this process, the molecular mechanisms that govern the regulation of ubiquitination remain largely unknown. Here, we have used a combination of yeast proteome chip assays, genetic screening, and in vitro/in vivo biochemical analyses to identify and characterize eight novel in vivo substrates of the ubiquitinating enzyme Rsp5, a homolog of the human ubiquitin-ligating enzyme Nedd4 in yeast. Our analysis of the effects of a deubiquitinating enzyme, Ubp2, has demonstrated that an accumulation of K63-linked poly-ubiquitin chains results in processed forms of two substrates, Sla1 and Ygr068c. Finally, we have shown that the localization of another newly identified substrate, Rnr2, is Rsp5-dependent. We believe that our approach constitutes a paradigm for the functional dissection of an enzyme with pleiotropic effects.