Although panduratin A, a chalcone derivative isolated from the rhizome of Kaempferia pandurata Roxb., represents a variety of pharmacological activities, no validated method for determination of panduratin A levels in biological samples is yet available. Thus, we developed a liquid chromatographic method using a tandem mass spectrometry for the determination of panduratin A in rat plasma. After simple protein precipitation with methanol including flufenamic acid (internal standard, IS), the analytes were chromatographed on a reversed-phased column with a mobile phase of distilled water and acetonitrile including 2% of formic acid (2:8, v/v). The ion transitions of the precursor to the product ion were principally deprotonated ions [M−H]− at m/z 405.2 → 165.9 for panduratin A and 280.1 → 236.0 for the IS. The accuracy and precision of the assay were in accordance with FDA regulations for the validation of bioanalytical methods. This analytical method was successfully applied to monitor the plasma concentrations of panduratin A following oral administration in rats.