Ubiquitination is a posttranslational modification in eukaryotic cells, where ubiquitin (Ub) is covalently conjugated to a protein substrate. Ub can form different polymeric chains by attaching Ub subunits to itself through its seven lysine residues or N-terminal methionine, forming a diversity of Ub chains on a protein substrate. The type of the chain modifying a substrate is known to correlate with the biological outcome of ubiquitination. However, even with some evidence present, it still poses a significant challenge to systematically assess the structure-function relationship. A major limitation is the lack of universal methods to characterize Ub conjugates. Mass spectrometry (MS)-based approaches are widely used in the characterization of Ub conjugates. With all the advantages of available MS-based workflows, there is still a challenge in capturing Ub chains with complex topologies and establishing their connectivity with a substrate. This thesis describes the development of novel MS-based tools for analyzing intact free or substrate-conjugated Ub chains. First, the thesis discusses the application of ion mobility-mass spectrometry (IM-MS) in the analysis of isomeric Ub chains. We developed an IM-MS-based deconvolution approach that enables the quantitation of the relative abundance of Ub isomers in a mixture. Additionally, by coupling IM-MS with collision-induced unfolding (CIU), we developed a deconvolution approach to assess the selectivity of deubiquitinating enzymes toward different isomeric Ub chains (DUBs) in a qualitative and time-dependent manner. Lastly, the thesis describes the application of top-down MS coupled with tandem MS (MS2) analysis in the characterization of Ub conjugates. We proposed a computational algorithm that enables the automatic annotation of MS2 fragmentation spectra and assigns probability scores for the analyzed Ub conjugates.