Protein ubiquitylation plays a pivotal role in most cellular pathways. The small protein ubiquitin (Ub) is covalently attached to other proteins through a series of enzymatic steps. Once a single Ub is anchored to target protein, subsequent rounds of conjugation can result in the formation of Ub chains. During chain extension, there are seven ε-amino groups (K6, K11, K27, K29, K33, K48, K63) along with an N-terminus (M1) that can be conjugated to the C-terminus of another Ub molecule. The diversity of Ub chains that manifests is astounding. The linkage, length, and degree of chain branching (i.e., how many Ub subunits are modified at more than one site with other Ub molecules) are all variables and each one can control the cellular destiny of the modified protein. Characterizing the types of chains attached to a protein-of-interest (POI) is thus critical to understanding how Ub regulates function, stability, and cellular localization. Mass spectrometry (MS) is the gold standard for characterizing Ub conjugates.