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A selective and sensitive LC–MS‐MS method was developed and validated for simultaneous estimation and pharmacokinetic studies of 16α‐hydroxycleroda‐3,13(14) Z‐dien‐15,16‐olide (K‐09) obtained from Polyalthia longifolia and its metabolite (K‐9T), a novel antidyslipidemic agent. Sample clean‐up involved liquid–liquid extraction of both the analytes and internal standard (rosuvastatin) from 200 μL of hamster plasma. The analytes were chromatographically separated on a Symmetry‐Shield C₁₈ (5 µm, 4.6 × 150 mm) column, using acetonitrile–0.1% aqueous formic acid (92:08, v/v) as the mobile phase. Detection was performed using negative ion electrospray ionization in multiple reaction monitoring mode. The MS/MS response was linear over the concentration range 1.56–200 ng/mL, with a correlation coefficient (r²) of 0.998 or better. The within‐ and between‐batch precisions (relative standard …