Deubiquitinases (DUBs) are essential regulators of protein homeostasis that influence cellular signaling, protein stability, and degradation by removing ubiquitin chains from substrate proteins. Understanding DUB–substrate interactions is critical to elucidate their functional roles and therapeutic potential. This review highlights key methodologies to investigate DUB activity and substrate interactions, including biochemical assays, fluorescence-based approaches, and in vitro deubiquitination assays. Biochemical methods, such as those measuring protein degradation rates, ubiquitination dynamics, and protein–protein interactions, provide valuable insights into DUB function and specificity. Fluorescence-based techniques that include photoconvertible reporters, fluorescent timers, and FRET enable the real-time monitoring of DUB dynamics and substrate turnover in live cells. Furthermore, in vitro deubiquitination assays provide direct mechanistic insights into DUB activity on target substrates. While each method provides unique insights, they also present challenges, like limited specificity or sensitivity, technical difficulties, or insufficient physiological relevance. Integrating complementary approaches can enhance accuracy and provide deeper insights into DUB–substrate interactions, facilitating the development of DUB-targeted therapeutic strategies.