A method is provided for determining in real time the course of thrombin activity in a sample of blood or plasma as it appears in and disappears from the sample which comprises adding a thrombin substrate to the sample that, per unit time, produces a detectable signal in a quantity that bears relation to the amount of thrombin present. Simultaneously, in a control sample of the same blood or plasma in which thrombin generation is not triggered, the activity of a standard preparation with invariable thrombin activity is measured. The exact molar amount of thrombin present at any moment is obtained by comparison of the activity measured in clotting blood and the simultaneously measured calibrator. The method is useful inter alia for diagnosing hyper- and hypo-coaguable states, either congenital, acquired or drug-induced in humans and animals. Also provided is a kit for use in this method.