Upon erosion and rupture of an atherosclerotic plaque, collagen and serotonin (5-hydroxytyramine [5-HT]) induce a process of simultaneous platelet aggregation and vasoconstriction. Simultaneous inhibition of these pathophysiological processes, attainable by 5-HT inhibition, is a potential drug target and could offer an attractive treatment modality. The availability of a reliable and accurate test to measure inhibition of 5-HT-induced platelet aggregation would facilitate the rational development of such new compounds. Therefore, we developed a validated method to measure the additive effect of 5-HT on platelet aggregation in human whole blood after an initial induction by a low-concentration collagen, using impedance aggregometry. This method is feasible to measure 5-HT-induced platelet aggregation in whole blood for the evaluation of promising platelet aggregation inhibitors possessing 5-HT antagonistic activity. The availability of this method will support and stimulate selective 5-HT antagonism as effective management of thrombosis.