Levonorgestrel (LNG) used in contraceptive methods, necessitates accurate pharmacokinetic (PK) profiling to ensure efficacy and safety. The objective of this study was to develop and validate a highly sensitive LC-MS/MS method for quantification of LNG in human serum and its application in a pharmacokinetic study. 500 µL sample was processed using a liquid-liquid extraction (LLE) protocol optimized for LNG recovery that yielded consistent analyte recovery while minimizing matrix interferences. Chromatographic separation was performed on Waters ACQUITY UPLC BEH C18 reverse-phase analytical column (2.1× 100 mm, 1.7 µm) using mobile phase consisting of acetonitrile and water with 0.1% formic acid (70:30, v/v), delivered under a gradient elution at 300 µL/min flow rate. These conditions provided sharp peak resolution and minimized run times. Mass spectrometric analysis was run with Waters XEVO TQ ABSOLUTE triple quadrupole instrument operating in positive electrospray ionization mode and monitored LNG using multiple reaction monitoring with precursor ion at m/z 312.98>81.468, 90.921, 108.963 amu. Optimized fragmentation parameters yielded high signal-to-noise ratios, enabling trace-level detection in biological matrices. The method demonstrated linearity over calibration ranges of 32.5–2000 pg/mL, with correlation coefficients (R²) exceeding 0.997. Precision and accuracy met bioanalytical method validation guidelines, confirming the robustness of assay for PKs. The validated method was successfully employed to measure serum LNG in a PK study among women implanted in Bangladesh. The method’s sensitivity facilitated LNG detection supporting assessment of drug-release performance in clinical settings. The method proved highly reliable, sensitive, and specific for quantifying LNG in human serum, supporting its use in both clinical monitoring and research applications.