Ubiquitin signaling requires tight control of all aspects of protein ubiquitination, including the timing, locale, extent, and type of modification. Dysregulation of any of these signaling features can lead to severe human disease. One key mode of regulation is through the controlled removal of the ubiquitin signal by dedicated families of proteases, termed deubiquitinases. In light of their key roles in signal regulation, deubiquitinases have become a recent focus for therapeutic intervention as a means to regulate protein abundance. This work and recent discoveries of novel deubiquitinases in humans, viruses, and bacteria, provide the impetus for this chapter on methods for evaluating the activities and structures of deubiquitinases. An array of available deubiquitinase substrates for biochemical characterization are presented and their limitations as standalone tools are discussed. Methods for the determination and analysis of deubiquitinase structure are also presented, with a focus on visualizing recognition of the ubiquitin substrate.