Tissue factor (TF) pathway inhibitor I (TFPI) is the physiological inhibitor of TF-induced blood coagulation. Circulating blood contains full-length TFPI and TFPI truncated at the C-terminal end. Previous studies have shown that full-length TFPI exerts a stronger anticoagulant effect on diluted prothrombin time (DPT) than truncated TFPI, and it has been suggested that full-length TFPI is biologically more important in vivo. The objective of this study was to develop and validate an assay of TFPI anticoagulant activity. TFPI anticoagulant activity was assayed using a modified DPT assay. Plasmas were incubated in the absence and the presence of TFPI-blocking antibodies. Results were expressed as a ratio with the clotting time in the presence of anti-TFPI as the denominator. The ratio was normalized against a ratio obtained with a reference plasma. The assay was compared with assays of TFPI free antigen, total antigen, and bound TFPI, and TFPI chromogenic substrate activity. We performed all tests in 436 healthy individuals. The normalized TFPI anticoagulant ratio was strongly associated with TFPI free antigen (r = 0.73) but was weakly associated with TFPI chromogenic substrate activity (r = 0.46), TFPI total antigen (r = 0.48), and bound TFPI (r = 0.30). TFPI chromogenic substrate activity was strongly associated with TFPI total antigen (r = 0.73). We have developed a novel assay of TFPI anticoagulant activity in plasma, which may be considered a functional assay of full-length TFPI. Further studies are needed to establish the role of TFPI anticoagulant activity for thrombotic disorders.