Abstract Platelets, or thrombocytes, are anucleated tiny blood cells with an indispensable contribution to the hemostatic properties of whole blood, detecting injured sites at the surface of blood vessels and forming blood clots. Here, we quantitatively and non-invasively investigated the morphological and biochemical alterations of individual platelets during activation in the absence of exogenous agents by employing 3-D quantitative phase imaging (QPI). By reconstructing 3-D refractive index (RI) tomograms of individual platelets, we investigated alterations in platelet activation before and after the administration of various platelet agonists. Our results showed that while the integrity of collagen-stimulated platelets was preserved despite the existence of a few degranulated platelets with developed pseudopods, platelets stimulated by thrombin or thrombin receptor-activating peptide (TRAP) exhibited significantly lower cellular concentration and dry mass than did resting platelets. Our work provides a means to systematically investigate drug-respondents of individual platelets in a label-free and quantitative manner, and open a new avenue to the study of the activation of platelets. Abstract Figure