The ProC Global is a clotting assay designed to globally evaluate the functionality of the protein C (PC) pathway, which is found defective in up to 30% of the Caucasian patients with thrombophilia. It is based on the ability of endogenous activated protein C (APC), generated by activation of PC by a snake venom extract, to prolong an activated partial thromboplastin time (APTT). This retrospective study was carried out to evaluate the ability of this assay to distinguish patients with or without any PC pathway abnormalities in a cohort of 899 unselected patients with a history of thrombosis. The result of the ProC Global assay, expressed in PC activation time-normalized ratio (PCAT-NR), was significantly lower in patients in whom was previously demonstrated an abnormality of the PC pathway compared to those without. The cut-off level of PCAT-NR=0.75 was found to provide the best sensitivity-specificity ratio, since all the patients with the factor V (FV) Leiden mutation (n=71), APC resistance (n=3), PC deficiency (n=22), or combined defects (n=19) had a PCAT-NR below that value. The sensitivity of the ProC Global assay for a low protein S (PS) level (n=56) was only 66%, and was even weaker in the case of hereditary PS deficiency (46.6%, n=15). The assay did not perform well in samples from patients on oral anticoagulant treatment (OAT, n=64) or with liver failure (n=4), as the PCAT-NR was reduced in most cases, even in the absence of any abnormality. These results suggest that the ProC Global assay could be validly used as a first-step screening test for the FV Leiden-related APC resistance and PC deficiency in patients not on OAT. Given the moderate sensitivity of the assay for PS deficiency, this coagulation inhibitor must be determined in every case. However, the overall benefit of such a screening strategy is limited since more than 38% of the 659 patients without abnormality had a decreased PCAT-NR.