A method is described which enables a quantitative measurement of the concentration of activated factor VIII (VIIIa) in plasma. Based on the ability of factor VIIIa to accelerate the activation of factor X by factor IXa, phospholipid and calcium ions, the course of factor X activation in time is measured using a chromogenic substrate. Free factor Xa is able to activate non-activated factor VIII present in a plasma sample, which increases the factor X activation velocity, and thus disturbs the measurement of factor VIIIa. Furthermore, factor Xa was found to be inactivated by serine protease inhibitors from the plasma sample. By adding surplus chromogenic substrate these reactions of factor Xa are inhibited and at the same time the rate of substrate conversion is a measure of the amount of factor Xa present. Factor X activation and amidolysis of chromogenic substrate then take place simultaneously. It is shown that under proper conditions the factor X activation velocity is linearly proportional to the factor VIIIa concentration. This causes the optical density to increase as a parabolic function of time. The concentration of factor VIIIa can be obtained from the quadratic coefficient of the equation describing the parabola. The method is specific for factor VIIIa in that the extrinsic factor X activator is shown to have no influence on the measurement of factor VIIIa in thromboplastin activated plasma. We conclude that a sensitive and reliable method for assessing factor VIIIa concentrations in plasma has been developed on the basis of simultaneous inhibition and measurement of factor Xa by a high concentration of chromogenic substrate.