While there are hundreds of predicted E3 ligases, characterizing their applications for targeted protein degradation has proved challenging. Here, we report a chemical biology approach to evaluate the ability of modified recombinant E3 ligase components to support neo-substrate degradation. Bypassing the need for specific E3 ligase binders, we use maleimide-thiol chemistry for covalent functionalization followed by E3 electroporation (COFFEE) in live cells. We demonstrate that electroporated recombinant von Hippel-Lindau (VHL) protein, covalently functionalized at its ligandable cysteine with JQ1 or dasatinib, induces degradation of BRD4 or tyrosine kinases, respectively. Furthermore, by applying COFFEE to SPSB2, a Cullin-RING ligase 5 receptor, as well as to SKP1, the adaptor protein for Cullin-RING ligase 1 F box (SCF) complexes, we validate this method as a powerful approach to define the activity of previously uncharacterized ubiquitin ligase components, and provide further evidence that not only E3 ligase receptors but also adaptors can be directly hijacked for neo-substrate degradation. Display omitted •COFFEE tests neo-substrate degradation by chemically modified E3 ligase components•Recombinant E3s are labeled with maleimide-functionalized neo-substrate ligands•Proof-of-concept degradation by electroporated JQ1- or dasatinib-labeled VHL•SPSB2 and SKP1 can be redirected to degrade tyrosine kinases Pinch et al. present a method to test neo-substrate degradation by recombinant E3 ligase components in live cells, called COFFEE (covalent functionalization followed by E3 electroporation). Following proof-of-concept studies with VHL, they demonstrate neo-substrate degradation by the Cullin-RING ligase receptor SPSB2, and by the adaptor protein, SKP1.