This study presents a systematic and quantitative assessment of the ubiquitin-modified proteome (ubiquitome) using a monoclonal antibody that recognizes diGly (diisoseroyl) isopeptides. The authors identified approximately 19,000 diGly-modified lysine residues in about 5,000 proteins. They monitored temporal changes in diGly site abundance in response to proteasomal and translational inhibition, revealing that ongoing translation is necessary for detecting alterations in site abundance. The study also demonstrated that quantitative diGly proteomics can identify substrates for cullin-RING ubiquitin ligases (CRLs). The analysis of the ubiquitome provides insights into alterations in protein homeostasis fidelity and the identification of CRL substrates. The findings highlight the importance of ubiquitylation in protein degradation and quality control, and suggest that the diGly-modified proteome may serve as a unique indicator of proteome health.This study presents a systematic and quantitative assessment of the ubiquitin-modified proteome (ubiquitome) using a monoclonal antibody that recognizes diGly (diisoseroyl) isopeptides. The authors identified approximately 19,000 diGly-modified lysine residues in about 5,000 proteins. They monitored temporal changes in diGly site abundance in response to proteasomal and translational inhibition, revealing that ongoing translation is necessary for detecting alterations in site abundance. The study also demonstrated that quantitative diGly proteomics can identify substrates for cullin-RING ubiquitin ligases (CRLs). The analysis of the ubiquitome provides insights into alterations in protein homeostasis fidelity and the identification of CRL substrates. The findings highlight the importance of ubiquitylation in protein degradation and quality control, and suggest that the diGly-modified proteome may serve as a unique indicator of proteome health.