Two simple and sensitive high performance liquid chromatography–tandem mass spectrometry(HPLC–MS/MS) methods were developed and validated for the determination of fenticonazole in human plasma after percutaneous and intravaginal administration. Mifepristone was used as an internal standard(IS), and simple protein precipitation by acetonitrile containing 2% acetic acid was utilized for extracting the analytes from the plasma samples. Chromatographic separation was performed on a Kinetex XB-C_(18) column. The quantitation was performed by a mass spectrometer equipped with an electrospray ionization source in multiple reactions monitoring(MRM) positive ion mode using precursor-to-product ion transitions of m/z 455.2–199.1 for fenticonazole and m/z 430.2–372.3 for mifepristone. The validated linear ranges of fenticonazole were 5–1000 pg/m L and 0.1–20 ng/m L in plasma for the methods A and B, respectively. For the two methods, the accuracy data ranged from 85% to 115%, the intra- and inter-batch precision data were less than 15%, the recovery data were more than 90%, and no matrix interference was observed. The methods A and B were successfully validated and applied to the pharmacokinetic studies of fenticonazole gel in Chinese healthy volunteers after percutaneous and intravaginal administration, respectively. Two simple and sensitive high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) methods were developed and validated for the determination of fenticonazole in human plasma after percutaneous and intravaginal administration. Mifepristone was used as an internal standard (IS), and simple protein precipitation was performed on a Kinetex XB-C (18) column. The quantitation was performed by a mass spectrometer equipped with an electrospray ionization source in multiple reactions monitoring (MRM) positive ion mode using precursor-to-product ion transitions of m / z 455.2-199.1 for fenticonazole and m / z 430.2-372.3 for mifepristone. The validated linear ranges of fenticonazole were 5-1000 pg / m L and 0.1-20 ng / mL in plasma for the methods A and B, respectively. For the two methods, the accuracy data ranged from 85% to 115%, the intr a- and inter-batch precision data were less than 15%, the recovery data were more than 90%, and no matrix interference was observed. The methods A and B were successfully validated and applied to the pharmacokinetic studies of fenticonazole gel in Chinese healthy volunteers after percutaneous and intravaginal administration, respectively.