Electrochemical potentials are essential for cellular life. For instance, cells generate and harness electrochemical gradients to drive a myriad of fundamental processes from nutrient uptake and ATP synthesis to neuronal transduction. To generate and maintain these gradients, all cellular membranes carefully regulate ionic fluxes using a broad array of transport proteins. For that reason, it is also extremely difficult to untangle specific ion transport pathways and link them to membrane potential variations in live cell studies. Conversely, synthetic membrane models, such as black lipid membranes and liposomes, are free of the structural complexity of cells and thus enable to isolate particular ion transport mechanisms and study them under tightly controlled conditions. Still, there is a lack of quantitative methods for correlating ionic fluxes to electrochemical gradient buildup in membrane models. Consequently, the use of these models as a tool for unravelling the coupling between ion transport and electrochemical gradients is limited. We developed a fluorescence-based approach for resolving the dynamic variation of membrane potential in response to ionic flux across giant unilamellar vesicles (GUVs). To gain maximal control over the size and membrane composition of these micron-sized liposomes, we developed an integrated microfluidic platform that is capable of high-throughput production and purification of monodispersed GUVs. By combining our microfluidic platform with quantitative fluorescence analysis, we determined the permeation rate of two biologically important electrolytes – protons (H+) and potassium ions (K+) – and were able to correlate their flux with electrochemical gradient accumulation across the lipid bilayer of single GUVs. Through applying similar analysis principles, we also determined the permeation rate of K+ across two archetypal ion channels, gramicidin A and outer membrane porin F (OmpF). We then showed that the translocation rate of H+ across gramicidin A is four orders of magnitude higher than that of K+ unlike in the case of OmpF where similar transport rates were evaluated for both ions.