To validate our assay, we aimed to obtain the EC50 values for known compounds and compare them with literature values. A library of six established ion transporters (1-6, as shown in Figure ) were synthesised as standards. This library includes anion transporters from various structural classes (such as ureas, thioureas, and squaramides) with reported EC50 values spanning three orders of magnitude (from 0.005 to 10 mol% with respect to lipid). The chloride-nitrate antiport activity of these transporters was previously assessed using ion-selective electrode assays. The compound library was initially screened using 96-well plates, and we conducted three Hill plots for each of the compounds 1-6 (Figure ). To evaluate the reproducibility and experimental error of our measurements, each Hill plot for a specific compound was performed with a different batch of vesicles on separate plates. The EC50 values obtained by this method correlated strongly with literature values (R² = 0.99).