Summary. Background: Human-activated protein C (APC) is a serine protease with anticoagulant, anti-inflammatory and cytoprotective functions. This feature renders APC to be a promising vascular-inflammatory biomarker.Objective: The aim of the present study was the development and validation of a technique that allows the measurement of APC plasma levels under practical laboratory conditions.Methods/patients: Based on the APC-binding ssDNA aptamer HS02-52G we developed an oligonucleotide-based enzyme capture assay (OECA) that quantifies aptamer-captured APC through hydrolysis rates of a fluorogenic peptide substrate. After optimization of pre-analytical conditions, plasma APC levels were measured in healthy individuals and patients undergoing hip replacement surgery.Results and conclusion: A combination of APC–OECA with an aprotinin-based quenching strategy allowed APC analysis with a limit of detection as low as 0.022 ± 0.005 ng mL−1 (0.39 ± 0.10 pmol L−1) and a limit of quantification of 0.116 ± 0.055 ng mL−1 (2.06 ± 0.98 pmol L−1). While APC plasma levels in healthy individuals fell below the quantifiable range of the APC–OECA platform, levels substantially increased in patients undergoing hip replacement surgery reaching peak values of up to 12 ng mL−1 (214 pmol L−1). When normalized to the amount of thrombin generated, interindividual variabilities in the APC generating capacity were observed. In general, with a turn-around time from blood sampling to generation of test results of < 7 h, the APC–OECA platform allows sensitive and rapid determination of circulating APC levels under pathological conditions.