Traditional Chinese medicine (TCM) is a complex system, which consists of numerous compounds with related mechanisms to maximize therapeutic efficacy with minimal adverse effects. Some new methods disclosing the contribution of these constituents as well as their relationship in the formula are necessary for elucidating the bio-active constituents and the working mechanisms of TCM. In this study, depletion of target components using preparative HPLC followed by antiplatelet and anticoagulation activities evaluation was first applied to investigate the roles of paeoniflorin and senkyunolide I in a well-known formula, SiWu decoction. The results showed that both paeoniflorin and senkyunolide I not only directly brought about some bio-activities, but also indirectly made the contribution to the total bio-activity reflection of SiWu decoction, especially the latter should deserve to be drawn attention to the research of complicated bio-active constituents of TCM or its formula. So, the significant and effective approach will be very useful for the elucidation of the contribution of each different chemical constituent to the bio-activity of a TCM formula. Furthermore, this study demonstrated the potential utilization of preparative HPLC in the research of TCM.