Early detection of platelet activation is important for the diagnosis and follow-up of several pathological conditions that primarily or secondarily involve platelets in their pathogenesis. The golden standard assay to detect thrombocyte activation is represented by the release of serotonin, classically performed by demanding methodologies, such as high-performance liquid chromatography, 14C-labelling and enzyme-linked immunosorbent assay (ELISA). We developed a non-radioactive method, based on individual cells, for the detection of serotonin content in activated and resting platelets by flow cytometry. The assay was standardized on cells activated by Ca2+ ionophore or by sera from patients with heparin-induced thrombocytopenia (HIT). Cells were identified by CD41a surface staining and their serotonin content measured by specific antiserotonin intracytoplasmic staining, while their activation was independently shown by annexin V binding. Cellular degranulation was detected by flow cytometry in all the cases that were also positive by standard ELISA. Moreover, multiparametric flow cytometry analysis revealed that, although virtually all activated cells bind annexin V, serotonin was released only by the platelet subset that downmodulates surface CD41a.