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Proteolysis Targeting Chimeras (PROTACs) have emerged as novel therapeutics to target the undruggable proteome. PROTACs are heterobifunctional small molecules that both simultaneously bind to a target protein and a ubiquitin (Ub) E3 ligase. This subsequently leads to ubiquitination and degradation of the target protein. The In vitro ubiquitination kit has been developed to establish a high throughput approach that can accurately predict PROTAC efficiency by monitoring the protein’s intrinsic ability to get ubiquitinated. We offer this kit for three E3 ubiquitin ligases: Cereblon, VHL, and HDM2 to monitor PROTAC mediated ubiquitination for target of choice.