In eukaryotic cells, short-lived, regulatory and misfolded or denatured proteins are degraded by the ubiquitin–proteasome system (UPS), a highly regulated mechanism including the active marking of proteins for proteasomal degradation by ubiquitin. The UPS is critical in regulating proteostasis and dysfunctioning of this system has been implicated in diseases such as cancer and neurodegenerative disorders. We have investigated the effects of proteasome malfunctioning in Drosophila by monitoring the global proteome and ubiquitinome using SILAC proteomics. The proteasome was inactivated by either chemical inhibitors or by dsRNA mediated knockdown of specific proteasome subunit constituents. Upon proteasome perturbation both the global proteome and the ubiquitinome were remodeled to a great extent, albeit that the overall impact on the ubiquitinome was much more dramatic. The abundances of ~10% of all observable proteins were increased as a result of both de novo synthesis and of accumulation of ubiquitinated proteins. In the ubiquitinome analysis ~14,000 diGly peptides were identified and most of these could also be quantified. The far majority of diGly peptides showed increased levels, indicating that the pool of ubiquitinated proteins is highly dynamic. Interestingly, because of the high level of detail the ubiquitination dynamics of individual proteins could be explored. Several examples (e.g., Rps3 and H2B) will be discussed, in which different target lysine residues show either increased or decreased ubiquitination on the same protein, suggesting the occurrence of simultaneous and possibly functionally different ubiquitination events. In conclusion, the combination of quantitative proteomic methods with dsRNA mediated knockdown of individual subunits of the 26S proteasome offers a powerful tool to study the dynamics of the (modified) proteome upon perturbation of the UPS. This strategy opens up new avenues for the dissection of the mode of action of this cellular machinery, both under proteostasis and proteotoxic conditions.