In this tutorial article, we describe the optical properties of lucigenin, the areas of its use, the experimental procedure for the lucigenin assay, problems commonly encountered when using the assay, and solutions to these problems. The standard lucigenin assay to study Cl -/NO3 -antiport will be described in detail, followed by a discussion of other variations on the lucigenin assay. Additionally, the use of the closely related probe SPBA (bis(3-sulfopropyl)-9,9'-biacridine) for anion transport studies will be demonstrated.