Altered serotonin (5-HT) signaling is implicated in several neuropsychiatric disorders, including depression, anxiety, obsessive–compulsive disorder, and autism. The 5-HT transporter (SERT) modulates 5-HT neurotransmission strength and duration. This is the first study using rotating disk electrode voltammetry (RDEV) to measure 5-HT clearance. SERT kinetics were measured in whole brain synaptosomes. Uptake kinetics of exogenous 5-HT were measured using glassy carbon electrodes rotated in 500μL glass chambers containing synaptosomes from SERT-knockout (−/−), heterozygous (+/−), or wild-type (+/+) mice. RDEV detected 5-HT concentrations of 5nM and higher. Initial velocities were kinetically resolved with K_m and V_max values of 99±35 standard error of regression (SER) nM and 181±11 SER fmol/(s×mg protein), respectively in wild-type synaptosomes. The method enables control over drug and chemical concentrations, facilitating interpretation of results. Results are compared in detail to other techniques used to measure SERT kinetics, including tritium labeled assays, chronoamperometry, and fast scan cyclic voltammetry. RDEV exhibits decreased 5-HT detection limits, decreased vulnerability to 5-HT oxidation products that reduce electrode sensitivity, and also overcomes diffusion limitations via forced convection by providing a continuous, kinetically resolved signal. Finally, RDEV distinguishes functional differences between genotypes, notably, between wild-type and heterozygous mice, an experimental problem with other experimental approaches.