AbstractPlatelets are the smallest cellular component in the peripheral circulation. Their primary role is maintenance of hemostasis. The evaluation of platelets by flow cytometry has proven beneficial in the investigation of many disease states, including inherited defects, cardiovascular disease, stroke, and many other inflammatory processes. In the absence of an added exogenous platelet agonist, the activation state of in vivo circulating platelets can be measured. Inclusion of an exogenous agonist in the assay enables analysis of the reactivity of circulating platelets in vitro. This unit describes protocols to evaluate platelet surface glycoproteins, platelet activation status, and platelet responsiveness to agonist. Detailed discussion of appropriate sample handling, reagent preparation, flow cytometric set‐up, and data interpretation are provided for three independent assays.