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Activation of platelets plays a crucial role in the pathophysiology of thrombosis and hemostasis. Blood test reflecting in vivo platelet activation is therefore potentially useful in evaluating patients with thrombosis. However, monitoring of platelet activation requires an anticoagulant and protocol ensures that platelets activation status is not altered artifactually in vitro. To investigate effect of anticoagulants on platelet activation, blood from healthy donors were separately collected in citrate theophylline adenosine dipyridamole (CTAD), CTAD plus citric acid, 3.2% sodium citrate and K3EDTA. Each sample was divided into two groups of experiments, with or without 4% paraformaldehyde (PFA) fixation. The spontaneous in vitro platelet activation was investigated by indirect immunofluorescencence and analyzed by flow cytometry using CD62P monoclonal antibody. The results showed that percentage of CD62P-expressing platelets was markedly low in fixed sample. With PFA fixation, blood sampled drawn in CTAD plus citric acid showed less spontaneous platelet activation (2.37 ฑ 0.72 %) than those in CTAD, 3.2% sodium citrate and K3EDTA (5.73 ฑ 1.84 %, 4.92 ฑ 2.09 %, 4.27 ฑ 1.86%), respectively. In present study, it was conclude that blood sample intended for measuring of in vivo platelet activation should be collected in CTAD plus citric acid together with PFA fixed. Bull Chiang Mai Assoc Med Sci 2009; 42: 102-111.