The development of more effective drugs requires knowledge of their bioavailability and binding efficacy directly in the native cellular environment. In-cell nuclear magnetic resonance (NMR) spectroscopy is a powerful tool for investigating ligand-target interactions directly in living cells. However, the target molecule may be NMR-invisible due to interactions with cellular components, while observing the ligand by 1H NMR is impractical due to the cellular background. Such limitations can be overcome by observing fluorinated ligands by 19F in-cell NMR as they bind to the intracellular target. Here we report a novel approach based on real-time in-cell 19F NMR that allows measuring ligand binding affinities in human cells by competition binding, using a fluorinated compound as a reference. The binding of a set of compounds toward Hsp90α was investigated. In principle, this approach could be applied to other pharmacologically relevant targets, thus aiding the design of more effective compounds in the early stages of drug development.