The interest in studying the transmembrane transport of carboxylates is founded on their essential role in diverse cellular processes, as well as the presence of this functional group in many active pharmaceutical ingredients (APIs). The fluorescence of lucigenin is quenched by carboxylates, which can be used to monitor the transport of carboxylates through phospholipid membranes, typically in an antiport process with nitrate. As the quenching of the fluorescence of lucigenin depends on the organic moiety of the carboxylate, it is crucial to record the emission spectra of lucigenin in the presence of the selected carboxylate before performing the transport experiments. For instance, aromatic carboxylates such as benzoate quench the lucigenin emission more than aliphatic carboxylates such as acetate (Figure ). The carboxylate transport experiments can be performed following the standard protocol for liposome preparation as described in Section 3.2. However, the lucigenin solution and salt solution must be prepared using a buffer at the selected pH value (such as 225 mM NaNO3, 5 mM HEPES, pH 7), to control the acid-based speciation of the carboxylate appropriately. To perform the transport experiment, the fluorescence intensity decrease is recorded after the anion pulse (25 mM RCOO -). It should be noted that spontaneous diffusion of carboxylates has been observed in liposomes prepared in NaNO3 solution in absence of a transporter. However, the emission intensity of lucigenin decreases significantly faster when an anionophore assists the transport of the carboxylate, as shown for transporter 3 at 1:1000 transporter:lipid ratio (Figure ). Measuring the pH of the liposome suspension before and after the anion pulse is recommended as it could be significantly affected by the addition of the carboxylate solution. Finally, some carboxylates have displayed unusual behaviour on the lucigenin emission (Figure ), which limits the employment of this assay for certain carboxylate compounds.