Significance In the proteasome, an essential protease of eukaryotes, the hexameric ATPases (Rpt1-Rpt6) inject ubiquitinated proteins into the proteolytic core particle. Individual Rpt proteins assemble into the hexameric Rpt ring through binding to specific chaperones: Nas2, Hsm3, Rpn14, and Nas6. Here, we show that Rpt ring assembly uses a ubiquitination-mediated control. Not4 ubiquitinates Rpt5 by competing against Nas2 first and then Hsm3 throughout Rpt ring assembly. Thus, Not4 selectively recognizes a Rpt ring that matures without these chaperones. Rpt5 ubiquitination blocks recruitment of Rpn1 ubiquitin receptor and Ubp6 deubiquitinase, halting proteasome assembly and its recognition of ubiquitinated substrates. Our findings reveal an assembly checkpoint where Not4 monitors chaperone actions during hexameric ATPase ring assembly, ensuring the accuracy of proteasome holoenzyme maturation.