Method for in vitro determination of the activity over time of at least one proteolytic enzyme in a biological sample, preferably a plasma sample or a whole blood sample, comprising determining over time the conversion of a fluorogenic substrate specific for said proteolytic enzyme, said method comprising the steps of: a) contacting the sample with at least one fluorogenic substrate for a proteolytic enzyme wherein said fluorogenic substrate has native fluorescence and measuring the native fluorescence F 0 , and contacting said sample with an agent triggering the generation of said proteolytic enzyme within the sample; b) generating a fluorescent signal over time curve by measuring, over time while said proteolytic enzyme is generated within the sample, the time course of at least one experimental fluorescence signal F exp .t emitted from the sample as a result of the release of a fluorescent group of the fluorogenic substrate specific for said proteolytic enzyme upon enzymatic action of the generated proteolytic enzyme on said fluorogenic substrate; c) measuring in the same or in a similar sample, F max the maximal fluorescence that is obtained when all substrate has been converted into fluorescent product; d) generating a normalized ratio over time curve by converting the values of F exp.t measured in step b) to values defined as the normalized ratio values R t according to the equation R t = (F exp.t - F 0 )/(F max -F 0 ) , using F 0 and F max as obtained in steps a, b and c; e) generating a proteolytic enzyme generation over time curve by applying the formula E t =dR t /dt.(S 0 +K m /(1-R t ))/(k cat ), where S 0 is the initial substrate concentration and K m and k cat are kinetic constants that govern the interaction between the proteolytic enzyme and the fluorogenic substrate. a) contacting the sample with at least one fluorogenic substrate for a proteolytic enzyme wherein said fluorogenic substrate has native fluorescence and measuring the native fluorescence F 0 , and contacting said sample with an agent triggering the generation of said proteolytic enzyme within the sample; b) generating a fluorescent signal over time curve by measuring, over time while said proteolytic enzyme is generated within the sample, the time course of at least one experimental fluorescence signal F exp .t emitted from the sample as a result of the release of a fluorescent group of the fluorogenic substrate specific for said proteolytic enzyme upon enzymatic action of the generated proteolytic enzyme on said fluorogenic substrate; c) measuring in the same or in a similar sample, F max the maximal fluorescence that is obtained when all substrate has been converted into fluorescent product; d) generating a normalized ratio over time curve by converting the values of F exp.t measured in step b) to values defined as the normalized ratio values R t according to the equation R t = (F exp.t - F 0 )/(F max -F 0 ) , using F 0 and F max as obtained in steps a, b and c; e) generating a proteolytic enzyme generation over time curve by applying the formula E t =dR t /dt.(S 0 +K m /(1-R t ))/(k cat ), where S 0 is the initial substrate concentration and K m and k cat are kinetic constants that govern the interaction between the proteolytic enzyme and the fluorogenic substrate. <EOS>