Even though platelets are known to play a critical role in hemostasis, mediated in part by their uptake, storage, and release of serotonin, there are many unexplored aspects of this process. Herein, single-cell amperometry is employed to characterize the dynamic secretion of serotonin from platelet dense-body granules. On the basis of a three-dimensional random walk simulation that estimates detection efficiency with varied spacing between the carbonfiber microelectrode and the platelet, it is clear that the detected charge likely represents complete oxidation of the released granule contents and, thus, is a good method to calculate the serotonin concentration in each granule. Using the measured charge and volume estimates based on transmission electron microscopy (TEM) data, the granular concentration of serotonin is approximately 0.5 M. The simulated spike widths are significantly narrower than most of the measured amperometric spikes, clearly indicating that the stored serotonin is highly associated with an aggregate rather than freely diffusible within the dense-body granule. Additionally, by varying extracellular buffer temperature and pH to adjust the driving forces for serotonin delivery from the dense-body granules to the extracellular space, it is clear that, although platelet chemical messenger storage and secretion is similar to that of other secretory cells, there are some important distinctions.