Background: Early diagnosis of immune heparin-induced thrombocytopenia (HIT) is essential to improve clinical outcome but remains challenging. The release of platelet microparticles (PMPs) is considered of major pathophysiological significance. Objectives: The aim of this study was to evaluate performances of PMP generation assay (PMPGA) compared to clinical outcome to diagnose HIT. The second objective was to compare PMPGA with performances of C-serotonin release assay (SRA) on the same series of patients. Methods: Sera of 53 HIT-suspected patients were retrospectively incubated with citrated-whole blood from healthy donors with 1 IU and 500 IU/ml of unfractionated heparin (UH). PMPGA was performed using FACSAria® flow cytometer. The clinical diagnosis was established by two blinded independent investigators analysing in a standardized manner the patient's medical records. Performances of PMPGA and SRA (n = 53) were evaluated using ROC curve analysis with clinical outcome as reference. Results: In positive HIT patients, PMPs expressing phosphatidylserine are generated with low UH concentration whereas PMP rate decreases significantly in presence of high UH concentration. Using clinical outcome as reference, sensitivity and specificity of PMPGA reached 88.9% (95% CI: 50.7-99.4) and 100.0% (95% CI: 90.0-100.0). Sensitivity and specificity of C-SRA were 88.9% (95% CI: 50.7-99.4) and 95.5% (95% CI: 83.3-99.2). Conclusions: PMPGA is a rapid and reliable assay for HIT diagnosis. PMPGA showed good correlation with C-SRA performances and predominately with clinical outcome.