Tight regulation of cell cycle progression is essential for the maintenance of genomic integrity in response to DNA injury. The aim of this study was to identify new deubiquitinating enzymes (DUBs) involved in the regulation of the G₂/M checkpoint. By using an siRNA-based screen to identify DUBs with an inherent ability to enhance a CDC25B-dependent G₂/M checkpoint bypass, we have identified 11 candidates whose invalidation compromises checkpoint stringency. We subsequently focused our attention on one of these, the previously uncharacterized USP50. Using a TAP-tag approach associated to mass spectrometry, in addition to a yeast-two-hybrid screen, we identified HSP90 as a major interacting partner for USP50. We also demonstrate USP50 depletion causes a loss in accumulation of the HSP90 client Wee1, which is an essential component of the G₂/M cell cycle arrest. Finally, we show that in response to DNA damaging agents, USP50 accumulates in the nucleus. We propose that USP50 may act through a HSP90-dependent mechanism to counteract CDC25B mitotic inducing activity and prevent Wee1 degradation, thereby repressing entry into mitosis following activation of the DNA damage checkpoint.