Modulation of protein activities by reversible post-translational modifications (PTMs) is a major molecular mechanism involved in the control of virtually all cellular processes. One of these PTMs is ubiquitination, which regulates key processes including protein degradation, cell cycle, DNA damage repair, and signal transduction. Because of its importance for numerous cellular functions, ubiquitination has become an intense topic of research in recent years, and proteomics tools have greatly facilitated the identification of many ubiquitination targets. Taking advantage of the StUbEx strategy for exchanging the endogenous ubiquitin with an epitope-tagged version, we created a modified system, StUbEx PLUS, which allows precise mapping of ubiquitination sites by mass spectrometry. Application of StUbEx PLUS to U2OS cells treated with proteasomal inhibitors resulted in the identification of 41 589 sites on 7762 proteins, which thereby revealed the ubiquitous nature of this PTM and demonstrated the utility of the approach for comprehensive ubiquitination studies at site-specific resolution.