A highly sensitive and specific HPLC–ESI-MS/MS method has been developed and validated for the estimation of centchroman with 100μL rat plasma using tamoxifen as an internal standard (IS). The assay procedure involved a single-step, liquid–liquid extraction of centchroman and IS from plasma with 2.5% (v/v) isopropanol in n-hexane, which yielded consistent recoveries of 109.5 and 107.8% for centchroman and IS in rat plasma, respectively. The total chromatographic run time was 3.8min. Peaks were resolved using 0.01M ammonium acetate (pH 4.5):acetonitrile (10:90, v/v) mobile phase on a Supelco Discovery C 18 column. Specificity and matrix effect on ionization was determined and found that method was specific and there was no significant matrix effect. Linearity range was found to be 0.5–100.0ng/mL with a correlation coefficient (r) of 0.9959 or better. The intra- and inter-day assay precision ranged from 3.3 to 9.0% and 5.5 to 6.8%, respectively, and intra- and inter-day assay accuracy was between 93.4–107.1% and 96.2–104.2%, respectively. Stability of centchroman in rat plasma was >89.0% in the battery of stability studies viz., bench-top, auto-sampler, freeze/thaw cycles and 30 days storage in a freezer at −80°C. The assay was successfully applied to determine the pharmacokinetic parameters in Sprague–Dawley rats after an oral administration of centchroman at 20mg/kg. As a result, the plasma half-life was 29.4±2.3h and the AUC (0–∞) was 7345.1±21.9ngh/mL. The maximum plasma concentration (C max ) 117.5±15.7ng/mL was achieved at 9.0±8.6h (t max ).