A facile activity assay for an H⁺-coupled transporter using florescent probes was developed with an H⁺-coupled manganese transporter (MntH) as a model. Making use of coupled-proton transport, the transport activity (H⁺/Mn²⁺ cotransport) can be directly determined via fluorescence intensity changes of the probe, 5-(and-6)-carboxyfluorescein (5(6)-FAM). The approach of using highly sensitive fluorescence probes provides a more simple and convenient assay method for the determination of proton-coupled metal-ion uptake by transporters.