The present invention relates to a method for analyzing the interaction between HIF-1 peptide and VBC protein using fluorescence polarization, more precisely, a method for quantitative analysis of formation of HIF-1-VBC protein complex which is composed of the steps of 1) preparing a fluorescent probe by attaching a fluorescein to hydroxyproline containing HIF-1 peptide; 2) reacting the fluorescent probe with VBC protein; and 3) measuring the fluorescence polarization of the above reactant and then comparing the fluorescence polarization with that of the fluorescent probe itself to investigate the changes of fluorescence polarization; a method for screening an inhibitor of the binding of HIF-1 peptide and VBC protein using the above method; and a method for analyzing the activity of prolyl hydroxylase using the above method. The method of the present invention enables simple analysis of the interaction between HIF-1 peptide and VBC protein by observing changes of fluorescence polarization and thus, it can be effectively used for high-speed screening using a well plate.