A small-molecule inhibitor of the deubiquitinating enzyme Usp14 enhances proteasome activity, thereby promoting the degradation of ubiquitin-protein conjugates. Usp14, a proteasome-associated deubiquitinating enzyme, inhibits the degradation of ubiquitin-protein conjugates by trimming the ubiquitin chain on the substrate. A catalytically inactive variant of Usp14 shows reduced inhibitory activity, suggesting that inhibition is mediated by trimming of the ubiquitin chain. A high-throughput screen identified a selective small-molecule inhibitor of Usp14's deubiquitinating activity. Treatment of cultured cells with this compound enhances the degradation of proteasome substrates implicated in neurodegenerative diseases. Usp14 inhibition accelerates the degradation of oxidized proteins and enhances resistance to oxidative stress. Enhanced proteasome activity through Usp14 inhibition may reduce levels of aberrant proteins under proteotoxic stress. The proteasome is essential for eukaryotic life and regulates many aspects of cell physiology. Most proteasome substrates are targeted via ubiquitination. The proteasome holoenzyme consists of a 19-subunit regulatory particle and a 28-subunit core particle. Substrate binding to the regulatory particle and translocation to the core particle for degradation occurs. Proteasome activity is regulated by numerous proteins that reversibly associate with it. Some bind the regulatory particle and deliver ubiquitin-conjugates to the proteasome, while others open the axial channel into the core particle. A third class of associated proteins, composed of ubiquitin ligases and deubiquitinating enzymes (DUBs), modifies proteasome-bound ubiquitin chains. Ubiquitin chains vary in linkage type and length, with longer variants interacting more strongly with the proteasome. Mammalian proteasomes are associated with three DUBs: Rpn11, Uch37, and Usp14. Uch37 and Usp14 reversibly associate with the proteasome, whereas Rpn11 is a stoichiometric subunit. These enzymes reside on the regulatory particle and remove ubiquitin from the substrate prior to degradation. The release of ubiquitin spares it from degradation, minimizing fluctuations in ubiquitin pools. The activity of Rpn11 on the substrate's ubiquitin chain is delayed until the proteasome is committed to degrading the substrate. Rpn11 then cuts at the base of a ubiquitin chain, freeing the substrate. Thus, removal of the ubiquitin chain by Rpn11 can promote substrate translocation into the core particle to be hydrolyzed. However, deubiquitination prior to commitment might inhibit substrate degradation, since ubiquitin targets the protein for degradation. In contrast to Rpn11, Usp14 and Uch37 can attack ubiquitin chains independently ofA small-molecule inhibitor of the deubiquitinating enzyme Usp14 enhances proteasome activity, thereby promoting the degradation of ubiquitin-protein conjugates. Usp14, a proteasome-associated deubiquitinating enzyme, inhibits the degradation of ubiquitin-protein conjugates by trimming the ubiquitin chain on the substrate. A catalytically inactive variant of Usp14 shows reduced inhibitory activity, suggesting that inhibition is mediated by trimming of the ubiquitin chain. A high-throughput screen identified a selective small-molecule inhibitor of Usp14's deubiquitinating activity. Treatment of cultured cells with this compound enhances the degradation of proteasome substrates implicated in neurodegenerative diseases. Usp14 inhibition accelerates the degradation of oxidized proteins and enhances resistance to oxidative stress. Enhanced proteasome activity through Usp14 inhibition may reduce levels of aberrant proteins under proteotoxic stress. The proteasome is essential for eukaryotic life and regulates many aspects of cell physiology. Most proteasome substrates are targeted via ubiquitination. The proteasome holoenzyme consists of a 19-subunit regulatory particle and a 28-subunit core particle. Substrate binding to the regulatory particle and translocation to the core particle for degradation occurs. Proteasome activity is regulated by numerous proteins that reversibly associate with it. Some bind the regulatory particle and deliver ubiquitin-conjugates to the proteasome, while others open the axial channel into the core particle. A third class of associated proteins, composed of ubiquitin ligases and deubiquitinating enzymes (DUBs), modifies proteasome-bound ubiquitin chains. Ubiquitin chains vary in linkage type and length, with longer variants interacting more strongly with the proteasome. Mammalian proteasomes are associated with three DUBs: Rpn11, Uch37, and Usp14. Uch37 and Usp14 reversibly associate with the proteasome, whereas Rpn11 is a stoichiometric subunit. These enzymes reside on the regulatory particle and remove ubiquitin from the substrate prior to degradation. The release of ubiquitin spares it from degradation, minimizing fluctuations in ubiquitin pools. The activity of Rpn11 on the substrate's ubiquitin chain is delayed until the proteasome is committed to degrading the substrate. Rpn11 then cuts at the base of a ubiquitin chain, freeing the substrate. Thus, removal of the ubiquitin chain by Rpn11 can promote substrate translocation into the core particle to be hydrolyzed. However, deubiquitination prior to commitment might inhibit substrate degradation, since ubiquitin targets the protein for degradation. In contrast to Rpn11, Usp14 and Uch37 can attack ubiquitin chains independently of