Targeted protein degradation (TPD) has risen as a promising therapeutic modality. Recently, we validated CUL3KLHL20 E3 ligase as a new actionable E3 ligase for TPD application by developing a synthetic macrocycle ligand BTR2000 to engage KLHL20. Linking the KLHL20 ligand to different target warheads, we have created PROTAC molecules to target various protein of interests. Since CUL3KLHL20 are localized in different sub-cellular compartments, here we report the first temporal and spatial characterization of CUL3KLHL20-driven TPD with BTR2000 derivative degrader molecules. Our study revealed the target protein degradation kinetics, PROTAC intracellular activity half-life, and the onset of the degrader cell permeabilization. Employing proximity ligation and confocal microscopy techniques, we also illustrate the sub-cellular location of the ternary complex assembly upon BTR2000 derived PROTAC treatment. These characterizations provide further insight into the processes that govern TPD and features that could be incorporated when designing future macrocyclic PROTAC molecules. I will also discuss the scope of target degradability of this new class of PROTAC-E3 ligase system.