Compounds 2 and 5 emerged as the most challenging ionophores to study within our series. For compound 5, the highest loadings (>0.02 mol% w.r.t. lipid) resulted in unexpectedly low transport activity (Figure ), necessitating the exclusion of these data points for fitting to the Hill equation. Our EC50 values for compound 5 were the least reproducible, with errors reaching up to 38%, while the largest discrepancy between literature and high-throughput (HT) EC50 values was observed for compound 2. We attribute these issues to difficulties in delivering these compounds to the vesicle bilayer. The low transport activity of compound 2 required testing at very high concentrations, which could have resulted in aggregation. Additionally, squaramides like compound 5 are poorly soluble in solvents other than DMSO due to the potential for intermolecular hydrogen bonding. Previous studies have indicated that more dilute stock solutions of compound 5 in DMSO exhibit higher transport activity, suggesting a challenge in delivering this compound effectively. While co-delivery with larger volumes of DMSO could enhance delivery into the bilayer, this approach is incompatible with our HT assay, which requires a constant DMSO volume to maintain consistent fluorescence quenching. Another significant distinction between our assay and the ion-selective electrode assay is the absence of stirring while adding the transporter solution. We hypothesise that the lack of stirring may exacerbate delivery issues. Further investigation of deliverability is critical for the potential biological applications, as some poorly deliverable ion transporters that demonstrate high activity in vesicle studies may show poor activity in cells. Overall, we have developed analogues of this assay with varying levels of automation. Figure illustrates the results from the manual, semi-automated, and fully automated HT methods, showing good agreement with literature values. The correlations between HT and literature EC50 values are visually represented, with R² values of 0.99, 0.98, and 0.89 for the manual, semi-automated, and automated methods, respectively. These strong correlations indicate that the HT assay produces accurate EC50 data comparable to that obtained through traditional methods. Our methods demonstrate significant potential to save time and enhance data generation within anion transport workflows. Using the manual 96-well method, we were able to prepare up to six plates in a single day, yielding a total of 18 Hill plots. In contrast, conventional procedures for generating Hill plots typically allow for only one plot to be collected per day. The use of a plate reader streamlines the process, enabling the addition of sodium chloride followed by fluorescence measurement to be completed in approximately 10 minutes per 96-well plate.