Publisher Summary The principle of the measurement of secretion of serotonin states that the radiolabeled serotonin that has been taken up equilibrates rapidly and totally with the endogenous serotonin and, therefore, behaves functionally as endogenous serotonin. This allows the quantitation of serotonin by a simple measurement of the total radioactivity. However, this ability also poses difficulties for secretion experiments because the secreted serotonin will rapidly re-enter the same platelets that secreted it. In the procedures outlined in this chapter, imipramine is added to a platelet suspension before secretion is induced. Alternatively, serotonin forms a fluorophore with O-phthalaldehyde (OPT), which can be easily detected down to 22 pmol under optimal fluorimetric conditions. The content of serotonin varies in platelets from different species, from 300 μmol/1011 human platelets to 6,930 μmol/1011 rabbit platelets. The OPT method described in the chapter is, therefore, suitable for human platelet suspensions of 1–3 × 108 cells/mL.