**Objective:** The aim of this study was to develop and validate a sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of medroxyprogesterone acetate in human plasma.
**Methods:** Plasma samples were spiked with an internal standard, megestrol acetate, and extracted using n-hexane. After evaporation under a nitrogen stream at 50°C, the residue was reconstituted in the mobile phase and analyzed by LC-MS/MS. The separation was performed on an Alltech Alltima C18 column (2.1 mm × 100 mm, 3 μm). The mobile phase consisted of methanol and 0.1% formic acid (72:28, v/v), delivered at a flow rate of 0.2 mL/min. The column temperature was maintained at 40°C. The electrospray ionization source operated in positive ion mode, and selective reaction monitoring (SRM) was used for detection. Quantitative analysis was based on the precursor-to-product ion transitions of m/z 327.4 for medroxyprogesterone acetate and m/z 325.4 for megestrol acetate.
**Results:** The calibration curve showed good linearity over the range of 0.1–8.0 μg/L, with a lower limit of quantification (LLOQ) of 0.1 μg/L. The method demonstrated high extraction recovery (76.1%) and good precision, with intra-day and inter-day RSD values below 9.0%.
**Conclusion:** This developed LC-MS/MS method is highly specific, sensitive, and easy to perform, making it suitable for routine clinical monitoring of medroxyprogesterone acetate in human plasma. The method provides reliable results and can be effectively applied in pharmacokinetic studies and therapeutic drug monitoring.
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