The neurotransmitter serotonin is important in mood and depression, acts as a vasoconstrictor in blood vessels and is the main neurotransmitter of the gut. In neurotransmission, the released serotonin is taken up by serotonin transporters, which are principal targets of antidepressants and the psychostimulant, ecstasy. Studies of serotonin transporters have relied almost exclusively on the use of radiolabeled serotonin in heterogenous end-point assays. Here we present the establishment and validation of the Serotonin (5-HT) Fluorescence Assay for Transport and Release (5-HT_FAsTR) for functional and pharmacological studies of serotonin transport and release. We demonstrate the applicability of the method for the study of a neuronal, high-affinity, low-capacity serotonin transporter (SERT) as well as an extraneuronal low-affinity, high-capacity organic cation transporter (OCT) and mutants thereof. 5HT_FAsTR is an accessible, versatile and reliable semi-homogenous assay format that only relies on a fluorescence plate reader for repeated, real-time measurements of serotonin influx and efflux.