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The COP9 signalosome (CSN) is a highly conserved eukaryotic multi-subunit enzyme, regulating cullin RING ligase activities and accordingly, substrate ubiquitination and degradation. We showed that the CSN complex of Saccharomyces cerevisiae that is deviated in subunit composition and in sequence homology harbors a highly conserved cullin deneddylase enzymatic core complex. We took advantage of the non-essentiality of the S. cerevisiae CSN-NEDD8/Rub1 axis, together with the enzyme-substrate cross-species activity, to develop a sensitive fluorescence readout assay, suitable for biochemical assessment of cullin deneddylation by CSNs from various origins. We also demonstrated that the yeast catalytic subunit, CSN5/Jab1, is targeted by an inhibitor that was selected for the human orthologue. Treatment of yeast by the inhibitor led to the accumulation of neddylated cullins and the formation of reactive oxygen species. Overall, our data revealed S. cerevisiae as a general platform that can be used for studies of CSN deneddylation and for testing the efficacy of selected CSN inhibitors.