In this work we evaluated the application of competitive ligand-observed F NMR experiments to estimate α of PROTAC induced ternary complexes (Figure ). A highly sensitive competitive F NMR assay based on spy molecule 19 (Scheme 1) was recently developed by us to detect small-molecules binding to the VHL E3 ligase. Given the multiple examples of VHL-recruiting PROTACs that form complexes with positive (α > 1), negative (α < 1) or no (α = 1) cooperativity, we decided to explore the main advantages and limitations of an NMR-based method to investigate these systems. All compounds shown are referred with the acronyms and numbers shown in their original citations to facilitate comparisons with reported data. Initially, a group of four VHL-binding PROTACs were chosen to benchmark the assay (Scheme 1). These compounds were previously shown to form ternary complexes with positive cooperativity (MZ1) or negative cooperativity (MZP-54, MZP-55 and MZP-61) with BET proteins. As with previous biophysical studies, a simplified version of the VHL E3 ligase (hereon referred as VBC) was employed in the assays, consisting of the VHL protein and adaptor subunits Elongin B and Elongin C. The 19 F CPMG spectra of spy molecule 19 (Scheme 1) in the absence or presence of the VBC were recorded, acting as controls for 100% and 0% displacement, respectively. Titrations of different concentrations of each PROTAC were performed to determine the concentrations at which 50% of the spy-protein complex was dissociated (IC50). These IC50 values were then converted to the respective inhibition constants (Ki). The same titrations were repeated in the presence of each bromodomain of the BET proteins Brd2, Brd3 and Brd4. Representative displacement curves are shown in Figure (complete set shown in ESI Figure ). .