Protein ubiquitination is a powerful post-translational modification implicated in many cellular processes. Although ubiquitination is associated with protein degradation, depending on the topology of polyubiquitin chains, protein ubiquitination is connected to non-degradative events in DNA damage response, cell cycle control, immune response, trafficking, intracellular localization, and vesicle fusion events. It has been shown that a ubiquitin chain can contain two or more topologies at the same time. These branched chains add another level of complexity to ubiquitin signaling, increasing its versatility and specificity. Mass spectrometry-based proteomics has been playing an important role in the identification of all types of ubiquitin chains and linkages. This review aims to provide an overview of ubiquitin chain topology and associated signaling pathways and discusses the MS-based proteomic methodologies used to determine such topologies. Significance: Ubiquitination plays important roles in many cellular processes. Proteins can be monoubiquitinated or polyubiquitinated forming non-branched or branched chains in a high number of possible combinations, each associated with different cellular processes. The detection and the topology of ubiquitin chains is thus of extreme importance in order to explain such processes. Advances in mass spectrometry based proteomics allowed for the discovery and topology mapping of many ubiquitin chains. This review revisits the state of the art in ubiquitin chain identification by mass spectrometry and gives an insight on the implication of such chains in many cellular processes.