A reliable and simple laboratory assay for predicting clinical effectiveness of antiplatelet acetylsalicylic acid (ASA) therapy is needed. We have compared various laboratory protocols for measuring blood thromboxane A2 (TXA2) generation used to detect the effects of ASA administration. Healthy volunteers (n = 15) were given 150 mg per day ASA for 10 days, followed by ASA at 75 mg per day for 10 days. Five protocols tested for measuring TXA2 generation were: baseline TXB2 determination in plasma; static generation of TXA2 in anticoagulated blood (1 h incubation at room temperature or 37°C, respectively); dynamic generation of TXA2 in anticoagulated blood (1 h in rotary mixer); and generation of TXA2 in blood without anticoagulant (serum-generated TXA2). Platelet aggregation in whole blood was also measured using arachidonic acid (AA), collagen, and ADP as agonists. All five protocols showed significant reduction in TXB2 levels in individuals taking ASA. However, only the assay of TXA2 generation in serum was significantly different compared with the other protocols (P < 0.002). Moreover, the strongest and most significant correlation was observed between TXA2 generation in serum and AA-induced aggregation parameters (for 75 mg per day ASA).Serum TXA2 generation is the best laboratory protocol to detect the effects of ASA, based on serum markers of prostanoid metabolism.