Bottom-up approaches are the most common, as proteolytic digestion breaks down Ub conjugates into peptide fragments, which are then analyzed by MS. Ubiquitylation sites are identified by a Ub remnant-a diGly motif-attached to the εamino group of a lysine or the α-amino group of an N-terminal residue. When combined with isotopically labeled peptide standards (i.e., AQUA peptides), this bottom-up approach enables quantitation of the absolute abundance of various Ub chain linkages. What bottom-up fails to provide is information on chain length and the extent of branching. These shortcomings have been addressed using topdown and middle-down MS approaches; however, quantitative information on linkage types is lost. By separating ions based on size and shape, ion mobility coupled with mass spectrometry (IM-MS) has the potential to inform on all aspects of Ub chains. IM is a measure of the time it takes an ion to drift through a buffer gas, with compact ions of similar charge traveling faster than those that are more elongated due to fewer interactions with the gas. Since Ub chains adopt different conformations depending on linkage, length, and degree of branching, IM-MS can, in principle, be used to separate and detect Ub chains of many different topologies present as heterogeneous mixtures. In line with this supposition, it was shown that several di-Ub isomers can be distinguished using drift tube IMS (DTIMS), and four di-Ub isomers can be distinguished using T-wave IM-MS by exploiting differences in the degree of unfolding as a function of linkage type. To further advance the use of IM-MS in Ub chain analysis, it is necessary to develop methods that enable relative quantitation of chain linkages in heterogeneous samples.