Synthetic anion transporters are developed to transport anions across lipid membranes with the long-term perspective of biological applications. The lucigenin assay is a popular tool to study their transport of chloride and other anions in liposomes. It relies on the quenching of the fluorescence of encapsulated lucigenin by anions, which can be monitored by fluorescence spectroscopy. This article provides a tutorial introduction to the practical use and understanding of the lucigenin assay. It describes in detail how to use this assay to monitor chloride/nitrate antiport in liposomes, process and interpret the data, and solve common issues. Variations of the assay enabling the investigation of the transport of other anions and transport mechanisms are discussed. Furthermore, a zwitterionic analogue of lucigenin is demonstrated to have advantages for use in experiments over longer time scales, as it does not leak out of the liposomes, or when studying chloride uniport, as it avoids interference from antiport with nitrate that is present in commercial lucigenin.