In-cell NMR spectroscopy is a unique approach to study the structure and function of biological macromolecules in their native cellular environment at atomic resolution. At CERM/CIRMMP, we developed an approach for expressing and labelling proteins directly in human cells, which is ideally applied to monitor functional processes such as protein folding and maturation, metal binding, chemical modifications, and interactions with ligands or with specific partners.1 A major limitation of in-cell NMR is the short lifetime of the cells once they are densely packed in a closed environment. We have implemented a modular NMR bioreactor to provide the cells with fresh nutrients and oxygen, which makes possible to study intracellular processes in real time over the course of up to 72 hours.2 Real-time in-cell NMR provides important information on protein-ligand interactions, such as intracellular ligand binding kinetics and thermodynamics, which are critical to optimize drug penetrance and potency.3 However, classical screening by 1H and 1H-15N spectra is limited by fact that signals from proteins interacting with cellular components are broadened beyond detection. In such cases, 19F NMR spectroscopy is an attractive alternative, thanks to the high-sensitivity and background-free nature of 19F. We have shown that fluorinated amino acids incorporated in proteins expressed in human cells allow protein-observed screening on otherwise invisible targets.4 In addition, fluorinated ligands can be directly observed as they interact with their intracellular targets. Furthermore, ligand-based in-cell screening can be achieved by competition binding, where the displacement of a fluorinated ligand is used to measure the binding affinity of a second non-fluorinated ligand. Such approaches hold great potential in the development of more effective drugs towards pharmacologically relevant targets.