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2’-Hydroxy-3’,4’-methylenedioxy-3,4,5-trimethoxychalcone (HMTC) is a newly synthesized chalcone that affects proliferation, cytotoxic potential and apoptosis in human leukemia cells. However, no validated determination method has been described so far for HMTC in biological samples. Thus, we developed a liquid chromatographic method using a tandem mass spectrometry to determine HMTC in rat plasma. Liquid-liquid extraction with ethyl acetate was used for the clean-up procedure. The analyte was separated on a reversedphase column with mobile phase of distilled water and acetonitrile (2:8, v/v, including 0.1 % formic acid). The ion transition of the precursor to the product ion was principally deprotonated ions [M-H]− at m/z 356.8 → 327.2 for HMTC. This analytical method was successfully applied in pharmacokinetic study of HMTC after intravenous administration in rats.