We raised a set of monoclonal antibodies (mAb) against recombinant human ADAMTS13, constructed the first antigen test fully based on mAbs and compared ADAMTS13 antigen and enzymatic activity levels in a large set of plasma samples collected from different patients and healthy controls. Assessing both ADAMTS13 antigen and activity helps to understand whether or not the protease is fully active in pathological conditions, e.g. liver cirrhosis, inflammatory bowel disease, cardiac surgery, pregnancy and oral contraceptive intake, in the neonatal state and in normal individuals. Our ADAMTS13 antigen assay showed less variability than the collagen binding-based activity assay. Antigen values correlated well with activity in normal individuals, but differed to various degrees in neonates, pregnancies at later maternal age and cardiac surgery. No discrepancies were noted in liver cirrhosis and inflammatory bowel disease, which were both associated with low plasma levels of ADAMTS13. In conclusion, parallel measurement of ADAMTS13 activity and antigen provides a new tool for understanding the behavior of the VWF-cleaving protease in health and disease.