The tyrosine kinase Wee1 is part of a key cellular sensing mechanism that signals completion of DNA replication, ensuring proper timing of entry into mitosis. Wee1 acts as an inhibitor of mitotic entry by phosphorylating cyclin-dependent kinase CDK1. Wee1 activity is mainly regulated at the protein level through its phosphorylation and subsequent degradation by the ubiquitin proteasome pathway. To facilitate identification of small molecules preventing Wee1 degradation, a homogeneous cell-based assay was developed using HeLa cells transiently transfected with a Wee1-luciferase fusion protein. To ensure ultra-high-throughput screening (uHTS) compatibility, the assay was scaled to a 1536-well plate format and cells were transfected in bulk and cryopreserved. This miniaturized homogeneous assay demonstrated robust performance, with a calculated Z′ factor of 0.65 +/− 0.05. The assay was screened against a publicly available library of approximately 218,000 compounds to identify Wee1 stabilizers. Nonselective, cytotoxic, and promiscuous compounds were rapidly triaged through the use of a similarly formatted counterscreen that measured stabilization of an N-cyclin B-luciferase fusion protein, as well as execution of viability assessment in the parental HeLa cell line. This screening campaign led to the discovery of 4 unrelated cell-permeable small molecules that showed selective Wee1-luciferase stabilization with micromolar potency. One of these compounds, SID-4243143 (ML118), was shown to inhibit cell cycle progression, underscoring the importance of Wee1 degradation to the cell cycle. This probe was found to be inactive in a whole-cell assay against its anti-target, cyclin B. In contrast, the current state-of-the-art probe, MG132, inhibits degradation of both Wee1 and cyclin B in the same assays. More importantly, flow-cytometry assays confirm that the probe is able to induce an increase in the G2/M population after cell treatment, without increasing the sub-G1 population, suggesting the probe is not toxic to cells. These results suggest that this uHTS approach is suitable for identifying selective chemical probes that prevent Wee1 degradation and generally applicable to discovering inhibitors of the ubiquitin proteasome pathway.