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PLK1 is a protein kinase that regulates mitosis and is both an important oncology drug target and a potential antitarget of drugs for the DNA damage response pathway or anti-infective host kinases. To expand the range of live cell NanoBRET target engagement assays to include PLK1, we developed an energy transfer probe based on the anilino-tetrahydropteridine chemotype found in several selective PLK inhibitors. Probe <b>11</b> was used to configure NanoBRET target engagement assays for PLK1, PLK2, and PLK3 and measure the potency of several known PLK inhibitors. In-cell target engagement for PLK1 was in good agreement with the reported cellular potency for the inhibition of cell proliferation. Probe <b>11</b> enabled the investigation of the promiscuity of adavosertib, which had been described as a dual PLK1/WEE1 inhibitor in biochemical assays. Live cell target engagement analysis of adavosertib via NanoBRET demonstrated PLK activity at micromolar concentrations but only selective engagement of WEE1 at clinically relevant doses.