The study investigates the role of Usp14, a proteasome-associated deubiquitinating enzyme, in regulating proteasome activity. Usp14 has been shown to inhibit the degradation of ubiquitin-protein conjugates both in vitro and in vivo, primarily through chain trimming of ubiquitin chains on substrates. A high-throughput screen identified a selective small-molecule inhibitor of Usp14, which enhanced the degradation of several proteasome substrates implicated in neurodegenerative diseases. The inhibitor, IU1, specifically targets Usp14 and does not affect other deubiquitinating enzymes. In cultured cells, IU1 reduced the accumulation of toxic proteins like Tau, TDP-43, and ataxin-3, and alleviated oxidative stress-induced cytotoxicity. These findings suggest that inhibiting Usp14 can enhance proteasome activity and potentially offer a strategy to reduce the levels of aberrant proteins in cells under proteotoxic stress.The study investigates the role of Usp14, a proteasome-associated deubiquitinating enzyme, in regulating proteasome activity. Usp14 has been shown to inhibit the degradation of ubiquitin-protein conjugates both in vitro and in vivo, primarily through chain trimming of ubiquitin chains on substrates. A high-throughput screen identified a selective small-molecule inhibitor of Usp14, which enhanced the degradation of several proteasome substrates implicated in neurodegenerative diseases. The inhibitor, IU1, specifically targets Usp14 and does not affect other deubiquitinating enzymes. In cultured cells, IU1 reduced the accumulation of toxic proteins like Tau, TDP-43, and ataxin-3, and alleviated oxidative stress-induced cytotoxicity. These findings suggest that inhibiting Usp14 can enhance proteasome activity and potentially offer a strategy to reduce the levels of aberrant proteins in cells under proteotoxic stress.