Ubiquitin signaling plays an essential role in controlling cellular processes in eukaryotes, and the impairment of ubiquitin regulation contributes to the pathogenesis of a wide range of human diseases. During the last decade, mass spectrometry-based proteomics has emerged as an indispensable approach for identifying the ubiquitinated proteome (ubiquitinome), ubiquitin modification sites, the linkages of complex ubiquitin chains, as well as the interactome of ubiquitin enzymes. In particular, implementation of quantitative strategies allows the detection of dynamic changes in the ubiquitinome, enhancing the ability to differentiate between function-relevant protein targets and false positives arising from biological and experimental variations. The profiling of total cell lysate and the ubiquitinated proteome in the same sets of samples has become a powerful tool, revealing a subset of substrates that are modulated by specific physiological and pathological conditions, such as gene mutations in ubiquitin signaling. This strategy is equally useful for dissecting the pathways of ubiquitin-like proteins.