Abstract: The measurement of binding kinetics is essential to optimize the efficacy and safety of therapeutic molecules and many studies have shown that the dissociation rate of antibodies is predictive for their clinical efficacy. Transmembrane proteins such as PD-(L)1, CD3, or HER2 are among the most studied drug targets, and it is known that their behavior and response to drug candidates are strongly influenced by folding and mobility conditions, as well as co-interactions with other membrane proteins. Retaining them within their native membrane environment enables the analysis of physiologically relevant interaction kinetics with high in vivo predictability. Despite of this, technologies for the automated measurement of real-time kinetic data on cells are currently unavailable. Here, we present Real-Time Interaction Cytometry (RT-IC), a novel technology that enables time-resolved measurements of kinetic rates and affinities on living cells. Measurements are performed with a heliXcyto biosensor instrument with advanced microfluidics. Workflows are fully automated with minimal hands-on time. RT-IC employs flow-permeable biocompatible cell traps for the automated immobilization of single cells on the surface of a biosensor chip. Adherent and suspension eukaryotic cells can be immobilized in a stable and reversible manner. Subsequently, the association and dissociation of fluorescently labeled analytes is measured in real-time. The applicability of RT-IC is demonstrated by examples including a broad spectrum of analytes, ranging from exosomes to small molecules. We show how difficult-to-isolate target proteins like GPCRs and ion channels may be investigated with RT-IC. Furthermore, we quantified the real-time binding kinetics of anti-CD3 and anti-PD-1 antibodies with their targets for the first time directly on T cells. Cell therapies may be advanced by the analysis of short-lived individual interactions like TCR-pMHC from the immunological synapse. The application data underlines the high value of RT-IC for drug discovery, as it provides kinetic interaction data that are relevant for physiological conditions and predictive for cellular processes in vivo.