Prolyl hydroxylase 2 (PHD2) is a key oxygen receptor regulating oxygen homeostasis in human body, and it is one of the important targets for drug research and development of hypoxia related diseases. In PHD2 enzymatic reaction, the structure of substrate (HIF-1 α556–574) and product (hydroxylated HIF-1 α) peptide only differ from one oxygen atom (MW > 20 0 0), which makes it a great challenge to separate them accu- rately and efficiently. In this work, the direct separation and detection of HIF-1 αand hydroxylated HIF- 1 αhas been firstly reported based on micellar electrokinetic chromatography (MEKC). Under optimized conditions, the intraday RSD of peak area and apparent electrophoretic mobility of hydroxylated HIF-1 αwere 1.87% and 0.81% respectively, and the interday RSD were 2.01% and 1.03% respectively. The LOD and LOQ of the MEKC method were 10 μM and 50 μM respectively, and the recoveries was 98.42–105.38%. Subsequently, the feasibility and accuracy of MEKC method to screen PHD2 inhibitors were confirmed by using roxadustat, and the IC ₅₀ (10.36 μM) and inhibitor type (competitive) were consistent with lit- erature. Finally, the method was used to screen the PHD2 inhibitory activity of five traditional Chinese medicines (TCMs). The present work not only overcomes the difficulties of direct quantitative detection of hydroxylated HIF-1 α, but also provides technical support for exploring and discovering new drug leads for hypoxia-related diseases from complex matrix such as TCMs.