Summary. Background: Protein S acts as a cofactor for full-length tissue factor pathway inhibitor (TFPI) in the downregulation of thrombin formation. Objective: To develop a functional test to measure the activity of the TFPI–protein S system in plasma. Methods/Patients: Using calibrated automated thrombography, we quantified the activity of the TFPI–protein S system in plasma by measuring thrombin generation in the absence and presence of neutralizing antibodies against protein S or TFPI. Moreover, we designed an enzyme-linked immunosorbent assay (ELISA) to determine the level of full-length TFPI in plasma. The performance of these assays was examined in plasma from 85 normal individuals and from 35 members of protein S-deficient families. Results: The ratio of thrombin peaks determined in the absence and presence of anti-protein S antibodies (protein S ratio = 0.5 in normal plasma) is a measure of the TFPI cofactor activity of protein S, whereas the ratio of thrombin peaks determined in the absence and presence of anti-TFPI antibodies (TFPI ratio = 0.25 in normal plasma) is a measure of the overall activity of the TFPI–protein S system. Protein S and TFPI ratios were elevated in protein S-deficient individuals, indicating an impairment of the TFPI–protein S system. Both ratios correlated well with full-length TFPI levels, which were significantly lower in protein S-deficient patients than in normal family members. Conclusions: Functional assays for the TFPI–protein S system and an ELISA for full-length TFPI were developed. These assays show that the activity of the TFPI–protein S anticoagulant pathway is impaired in individuals with congenital protein S deficiency.