The automation process was implemented in stages. Initially, we created a semi-automated protocol that allowed the Opentrons to dispense pre-mixed lipid and transporter solutions into the 384-well plate. Subsequently, we developed a fully automated method where the Opentrons performed all liquid handling steps, including diluting transporter stock solutions in DMSO, mixing the transporter solutions with the lipid, and dispensing these mixtures into the 384-well plates. Detailed standard operating procedures for each method, along with relevant instrument settings and data analysis spreadsheets, are available in the supplementary materials. The manual 96-well plate method exhibited the greatest variation in results, likely due to inconsistencies in manual pipetting and batch-to-batch variability among the vesicle samples. The standard deviations of the three EC50 values derived from replicate Hill plots for each compound ranged from 8% to 37% of the mean value (Figure ). While repeat Hill plots are infrequently reported in the literature, these error margins align with the variability observed in a large compound library assessed using ion-selective electrode assays, which reported errors between 9% and 38%. These findings underscore the inherent variability in EC50 measurements across experiments, highlighting the necessity for efficient protocols to facilitate repeat experiments.