Detection of reduced aspirin effectiveness has gained significant importance since clinical consequences of aspirin resistance were reported. Nevertheless, due to differentiated molecular basis of aspirin resistance, the conflicting choice of referential method for detection of acetylsalicylic acid ineffectiveness has become troublesome. This study, using a rat model of antiplatelet therapy, examines the aptitude of selected TXB2 metabolism-based methods in the detection of acetylsalicylic acid effectiveness. We hypothesized that ex-vivo whole blood spontaneous TXB2 generation assay could be, contrary to basal TXB2 and urine 11-dTXB2, a novel surrogate measure for impaired acetylsalicylic acid-dependent inhibition of thromboxane synthesis. To address this hypothesis, we evaluated the sensitivity of TXB2 generation assay in hirudinized whole blood to detect acetylsalicylic acid-mediated inhibition of cyclooxygenase activity in healthy rats and diabetic rats treated with acetylsalicylic acid. In diabetic and control animals, both acetylsalicylic acid drenches in the dose-independent manner contributed to significant attenuation of basal plasma TXB2 and urinary 11-dTXB2 formation. Urinary concentrations of 11-dTXB2 were, contrary to basal TXB2, significantly higher, regardless of acetylsalicylic acid dose, among all diabetic groups, compared with corresponding control groups. Determination of TXB2 generation in whole blood enabled sensitive detection of dose-related acetylsalicylic acid effect in both groups, as well as increased TXB2 formation in diabetes. We showed for the first time that evaluation of spontaneous generation of TXB2 in hirudinized whole blood enables, contrary to basal plasma TXB2 and urine 11-dTXB2 concentrations, to sensitively determine the acetylsalicylic acid effect in healthy and diabetic subjects.