摘要： Background: Progesterone is one of the female steroid hormones and plays an important role in the menstrual cycle and during pregnancy. It is especially important in preparing the uterus for the implantation of the blastocyst and maintaining pregnancy. The concentration in human serum is measured to determine the ovarian function retroactively and the cause of abortion in early pregnancy. Methods: A quantification assay based on isotope dilution mass spectrometry to determine the concentration of progesterone in human serum is reported. Incorporated with 13C3-progesterone, serum samples were subjected to progesterone extraction and clean-up by C4 solid-phase-extraction columns and hexane-based liquid/liquid extraction, respectively. The cleaned-up serum samples were then subjected to MALDI-TOF mass spectrometry for the quantification of progesterone. Results: Progesterone and the internal standard, 13C3-progesterone, were measured in the selected reaction monitoring mode for the transitions m/z 315.4 to 108.9 and m/z 318.4 to 111.9, respectively. We calculated the peak area ratio of progesterone to 13C3-progesterone in the mass spectrometry. The progesterone concentration in human serum was calculated by substituting the peak area ratio into an isotope dilution calibration curve, and then compared with the radioimmunoassay. Conclusions: In the study, the concentrations of serum progesterone were measured, and the recovered progesterone concentration determined by the assay showed good robustness and consistency in comparison to the conventional radioimmunologic assay. We concluded that the 13C3-progesterone-based quantification assay is a robust method for the measurement of serum progesterone.