In summary, we describe the application of a displacement NMR assay to interrogate the PROTAC:protein binding to form binary and ternary complexes. The advantage of the assay is that it leverages the power, sensitivity and speed of 19 F ligandobserved NMR spectroscopy to enable rapid monitoring of the cooperativity of PROTAC ternary complexes. Our study highlights both the potential and limitations of the assay for accurately estimating cooperativity. The assay robustly differentiated between positively and negatively cooperative PROTACs for a given target. While negative cooperativities were accurately estimated, the large positive cooperativity of MZ1 were underestimated, due to the tight binding regime found under the assay conditions. More accurate estimations were obtained when studying a cooperative PROTAC with weaker binary binding affinity to start from, and homo-PROTAC CM11 that cooperatively dimerizes its E3 ligase. The relatively high protein concentrations required for ligand-observed NMR and the presence of off-target interactions were identified as factors that can hinder assay development. Particular attention should be given to potential off-target interactions between the spy molecule and its non-cognate partner, guaranteeing that they are minimal enough to not interfere in the measurements. Due to the high sensitivity of ligand-observed F NMR to intermediate and weak binding affinities, this evaluation can be quickly performed during assay development with different targets. Despite these caveats, the described assay rapidly allowed at least semi-quantitative estimates of PROTAC cooperativities. We anticipate that this assay would prove beneficial to aid screening and characterization of PROTACs in medicinal chemistry discovery and optimization campaigns. Further work will be directed at extending the breadth of applicability to additional E3 ligases e.g. CRBN, and to monitoring cooperativity at the target protein end.