The present invention provides a process for the specific determination of the cholesterol of the HDL fraction in the presence of the LDL fraction of serum lipoproteins. Pancreatic cholesterol esterase is used to liberate cholesterol, and the liberated cholesterol then reacts with cholesterol oxidase and oxygen to form hydrogen peroxide. The kinetics of either of hydrogen peroxide formation or oxygen consumption is measured within 2 to 15 minutes after the start of the reaction between cholesterol and the oxidase. The temperature is maintained within a range of 20.degree. C., during a predetermined time interval. Specific concentrations of reactants are maintained in the reaction solution, i.e., from 0.05 to 30 U/ml pancreatic cholesterol esterase; from 0.1 to 50 U/ml cholesterol oxidase; from 1.0 to 20 mMole/liter of a tenside of the bile acid group, and 0.1 to 10 g/liter of a non-ionic detergent. The pH is kept within a range of 5 to 9. In addition, a reagent is provided which is used for specific determination of HDL fraction cholesterol in the presence of the LDL fraction of serum lipoproteins. The reagent contains pancreatic esterase (0.05 to 30 U/ml); cholesterol oxidase (0.1 to 50 U/ml) a tenside of the bile acid grop (1.5 to 8 mMole/liter) and a non-ionic detergent (0.1 to 10 g/liter), all concentrations referring to the dilution used in the test. The reagent also contains a buffer at a pH of from 5 to 9, and a system for photometric determination of hydrogen peroxide.