AbstractDirect-to-biology (D2B) synthesis and assaying has the potential to cut drug-discovery cycle times by providing information rich experiments in a highly efficient manner. For PROTACs, this cycle time is often slower than conventional medicinal chemistry as there are a plethora of small molecule E3-ligase or protein-of-interest binders and vectors to investigate as well as a vast landscape of linker chemical space to explore; finding the optimum for these three individual components is time-consuming and resource intensive. An attractive alternative would be to conduct miniaturized reactions in 1536-well plates, where all three PROTAC components are simultaneously evaluated, and directly assessed as crude reactions in cell-based HiBiT assays. Judicious reagents choice is required to ensure high reaction conversion and, moreover, minimize any effect on cell-viability. This talk will showcase our Direct-to-biology protocol to assess crude PROTACs using two well estabished PROTAC target case-studies: HER2 and BRD4.