To establish a high-throughput (HT) Hill plot assay in a 96-well plate format, we made several adaptations to existing protocols. While the traditional lucigenin assay continuously records fluorescence, the Hill plot only requires an end-point reading; thus, we focused on optimising the accuracy of this single measurement, which was taken five minutes after NaCl addition. Another key objective was to develop an assay compatible with DMSO as a delivery solvent for the anion transporter. DMSO is widely used in other ion transport assays due to its ability to dissolve a broad range of poorly soluble transporters, but it is rarely incorporated into the lucigenin assay because it is known to quench lucigenin fluorescence. Our investigations revealed that the assay could tolerate small amounts of DMSO (as long as the concentration was consistent across wells), with fluorescence partially quenched at 1% v/v. Notably, an intravesicular chloride concentration of 100 mM resulted in a six-fold fluorescence quenching response, even in the presence of 1% DMSO, and the Stern-Volmer plot remained linear from 0-100 mM [Cl⁻] under assay conditions (Supplementary Figure ). Therefore, by carefully controlling the DMSO concentration and the ionic gradients, we successfully utilised DMSO as a delivery solvent for our transporters.