INTRODUCTION: Covalent modification by ubiquitin via Lys isopeptide bonds is fundamental for regulating protein turnover and function. Additionally, ubiquitin esterification occurs on Ser/Thr/Tyr residues in proteins and on non-proteinaceous substrates including ribose, saccharides, lipids, and small molecule drugs. Ubiquitin posttranslational modifications may therefore be much more widespread across cell biological pathways. Recent literature (PubMed) reflects the increased interest in analytical methods for mapping of non-canonical substrates modified by ubiquitin and ubiquitin-like (UBL) proteins. AREAS COVERED: Mass spectrometry (MS)-based methodologies involve advanced proteomic techniques to identify ubiquitin modifications on amino acids other than Lys, such as Ser, Thr, Tyr and Cys as well as protein N-termini. After digestion, standard MS workflows identify canonical ubiquitination by detecting a ubiquitin C-terminal tag attached to the amine side chains of Lys residues of substrate-derived peptides suitable for MS/MS sequencing. For non-canonical modifications on proteins and substrates other than proteins, specialized strategies are required, such as using antibodies to enrich N-terminally modified peptides in combination with using high-resolution MS/MS based on softer fragmentation technologies to detect esterification and possibly other types of substrate modifications. EXPERT OPINION: Enabling such technologies will reveal a previously unrecognized angle of the ubiquitin code's complexity in cells.