Updated project metadata. We describe a modified workflow to enrich for and detect diGly peptides originating from ubiquitinated proteins using immunopurification. Using a combination of several relatively simple modifications in the sample preparation and mass spectrometric detection protocols, we now routinely detect over 24,000 diGly modified peptides in a single sample. We show the efficacy of this strategy for cell lysates from both non-labeled and metabolically labeled (SILAC) mammalian cells. Furthermore, we demonstrate that this optimized strategy is also useful for the in-depth identification of the endogenous, unstimulated ubiquitinome of in vivo samples such as mouse brain tissue. As such, this study presents an addition to the toolbox of the ubiquitination site analysis for the identification of the deep ubiquitinome.