This study established an HPLC-MS/MS method for qualitative and quantitative analysis of mifepristone and N-demethylmifepristone in blood samples. The appropriate pretreatment methods were investigated to pretreat the blood samples. HPLC was applied for separation. MRM mode was adopted to analyze. The precision, matrix effect, detection limit, recovery rate, and linear relationship of the method were all tested. The results indicated that mifepristone and N-demethylmifepristone in blood were separated well, with good chromatographic peak shape and good linear relationship in the range of 10 to 200 ng/mL. The detection limit concentration of mifepristone in blood was 0.5 ng/mL, the lower limit of quantification was 1.5ng/mL, the matrix effect was 94.6% to 98.2%, and the recovery rate was 82.7% to 92.4%; The detection limit concentration of N-demethylmifepristone was 1ng/mL, the lower limit of quantification was 2 ng/mL, the matrix effect was 91.2% to 97.3%, and the recovery rate was 81.8% to 91.7%. This method is simple, efficient, stable and easy to operate. It demonstrates satisfactory results for both qualitative and quantitative tests of mifepristone and N-demethylmifepristone, making it suitable for applying to the daily inspection and identification of related cases.