The generation of action potentials in neuronal cells and many other physiological processes involve the transport of chloride ions. Whilst there have been advances in chloride imaging techniques utilizing FRET biosensors, there is a lack of methodologies that are amenable to high-throughput screening for drug discovery. In this study, we developed a novel BRET-based biosensor (Glorider), utilizing a chloride-sensitive GFP variant fused to NanoLuciferase. The Glorider biosensor was then used to kinetically measure the effect of WNK, KCC2 and NKCC1 modulators in real time in living cells, including recently reported KCC2 agonists.