Ion transport is typically measured using ion-selective electrode assays or fluorescence assays, and we determined that the latter would be more suitable for adaptation to a high-throughput format. The lucigenin assay, a well-established method in the field, quantifies direct chloride transport across synthetic vesicle membranes, which are usually composed of 1-palmitoyl-2-oleoyl-glycero-3phosphocholine (POPC) or POPC/cholesterol mixtures. These vesicles contain NaNO3 and lucigenin (N,N'-dimethyl-9,9'-biacridiniumdinitrate), a fluorescent sensor that is quenched by chloride ions in a concentration-dependent manner, as described by the Stern-Volmer equation (equation S1). The assay begins with the addition of NaCl to the external buffer of the vesicles, allowing the anion transporter to facilitate passive chloride influx via a Cl⁻/NO3⁻ antiport mechanism, leading to fluorescence quenching. The fluorescence intensity can be calibrated against reference measurements; typically, 0% influx corresponds to the initial fluorescence shortly after the NaCl spike, while 100% influx is represented by the fluorescence reading obtained after adding detergent or a highly effective transporter. Various data processing methods can then be employed to quantify transport activity, such as calculating the EC50 or the specific initial rate. The EC50 is commonly reported as a measure of transport activity. It reflects the theoretical concentration of an anionophore required to achieve 50% ion influx or efflux by the end of the assay. This value is determined from a Hill plot, which is created by plotting the final chloride influx against transporter concentration. Traditionally, these experiments are performed individually in a cuvette or single vial, typically testing 6-8 different transporter concentrations in triplicate. As a result, this standard protocol usually allows for the assessment of only one compound per day due to time limitations.