Nonoxynol-9 (N-9), a microbicidal spermicide, has been in use as an over-the-counter contraceptive since the 1960s. A detailed account of its pharmacokinetic profile using highly sensitive detection method has not been reported yet. We developed and validated a rapid, selective and sensitive high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method for N-9 detection in plasma and simulated vaginal fluid. The analytes were quantified using reverse phase Thermo Accucore C18 (150 × 4.6 mm, 5 μm) column with isocratic elution using acetonitrile: 0.1% formic acid in triple distilled water (90:10, v/v) as mobile phase. The ionization was optimized using ESI (+) and selectivity was achieved by tandem mass spectrometric analysis using MRM transition, m/z 617.4→133.2 for N-9 and m/z 180.1→138.1 for phenacetin. The method was linear over the range of 0.195–100 ng/mL. The method was accurate and precise with intra‐batch and inter‐batch accuracy (% bias) of < ± 15% and precision (% CV) of < 15% for N-9. The mean peak plasma concentration (Cmax) 4.87 ± 0.37 ng/mL was achieved 1.0 hr after vaginal application with terminal half-life 1.45 ± 0.07 hr in rabbits. The validated method was successfully applied for pharmacokinetic study of N-9 in rabbits after vaginal administration