Ubiquitination is a reversible post-translational modification that maintains cellular homeostasis and regulates protein turnover. Deubiquitinases (DUBs) are a large family of proteases that catalyze the removal of ubiquitin (Ub) along with the dismantling and editing of Ub chains. Assessing the activity and selectivity of DUBs is critical for defining physiological functions. Despite numerous methods for evaluating DUB activity, none are capable of assessing activity and selectivity in the context of multicomponent mixtures of native unlabeled Ub conjugates. Here, we report an ion mobility (IM)-based approach for measuring DUB selectivity in the context of unlabeled mixtures of Ub chains. We show that IM-mass spectrometry (IM-MS) can be used to assess the selectivity of DUBs in a time-dependent manner. Moreover, using the branched Ub chain selective DUB UCH37/UCHL5 along with a mixture of Ub trimers, a strong preference for branched Ub trimers bearing K6 and K48 linkages is revealed. Our results demonstrate that IM-MS is a powerful method for evaluating DUB selectivity under conditions more physiologically relevant than single-component mixtures.