Abstract: A method for the absolute quantification of human leptin was established by high performance liquid chromatography-isotope dilution mass spectrometry （HPLC-IDMS）. The hydrolysis conditions optimized were 40 h with 6 mol/L HCl at 110 ℃. After hydrolysis, the sample was separated by HPLC, the transitions of proline (m/z 116＞m/z 70) and praline-C₅ ( m/z 121＞m/z 74), valine (m/z 118＞m/z 72) and valine-C₅ (m/z 123＞m/z 76), leucine (m/z 132＞m/z 86) and leucine-D₁₀ (m/z 142＞m/z 96) were monitored for quantitation by MRM mode. The content of human leptin was calculated to be 0.582 g/g with the uncertainty of 0.014 g/g（k=2）. The calculated results are further validated by the purity deduction assay. It shows good consistency between the results of the two methods. In conclusion, application of this method for quantification of leptin will improve the precision, accuracy and lay the foundation for the future research of reference material of leptin.