Von Hippel–Lindau (VHL) is a component of an E3 ubiquitin ligase complex that plays an important role in targeted protein degradation through the ubiquitin-proteasome pathway. The VHL complex consists of VHL, Cullin 2 (CUL2), Elongin B, Elongin C, and RBX1, and is widely used as an E3 ligase recruiter in PROTAC (Proteolysis-Targeting Chimera) drug discovery. VHL enables selective degradation of target proteins by recruiting them to the cellular ubiquitination machinery. The TR-FRET VHL Binding Assay Kit is designed to quantitatively measure the binding interaction between VHL and VHL ligands. In this assay, binding of fluorescently labeled VH032 ligand to Tag1-VHL-5C generates a fluorescence resonance energy transfer (FRET) signal detected by measuring fluorescence emission at 665 nm and 620 nm. Compounds that compete with VH032 for VHL binding reduce the HTRF signal, enabling evaluation of VHL-targeting molecules for drug discovery applications.