Cell cycle progression and entry into mitosis are regulated by a highly conserved cellular process known as checkpoint signaling. The Wee1 nuclear tyrosine kinase functions in this process by regulating the cdc2/cyclinB protein complex. Specifically, Wee1 mediates inhibitory phosphorylation of cdc2, leading to delayed mitosis and cell cycle arrest in cells with DNA damage so that DNA repair and replication can occur. Wee1 activity is inhibited during mitosis by its phosphorylation and ubiquitination by E3 ligases, and its subsequent degradation by the proteasome. Studies showing that Wee1 expression is reduced in colon carcinoma cells and that Wee1 overexpression can block cell division, suggest that Wee1 may act as a tumor suppressor. Thus, the identification of probes that selectively increase levels of Wee1 may provide useful insights into the roles of Wee1 in cell cycle control and tumor pathogenesis. The Scripps Research Institute Molecular Screening Center (SRIMSC), part of the Molecular Libraries Probe Production Centers Network (MLPCN), utilized a chemical biology approach to uncover kinases controlling Wee1 proteolysis by identifying small molecule inhibitors stabilizing a reporter of Wee1 degradation, K328M-Wee1-luciferase. High-throughput screening followed by medicinal chemistry optimization driven by secondary assays yielded a selective stabilizing probe, ML177, which is 200 times more potent than our previously described probe, ML118. ML177 exhibits a Wee1 stabilization EC50 value of 40 nM. The probe has been tested against several cell-cycle related targets and anti-targets, including Wee1, cyclin B, p21, p27, and ability to induce HeLa (cancer) cell apoptosis. The identified probe exhibited significantly improved selectivity and potency in these assays compared to the previous probe. These results, combined with additional kinase profiling by the assay provider, confirm the value of this probe and its usefulness in biologically relevant, cell-based assays.