Abstract: 5,6,7,4′-Tetrahydroxy-flavone and 5,6,7,4′-tetramethoxy-flavone were subjected to high resolution electrospray ionization tandem mass spectrometry (HR-ESI-MS/MS) in positive ion mode to obtain their protonated molecules and subsequent product ions, which were further analyzed to give proposed fragmentation pathways for the two flavones of polyhydroxy and polymethoxy substitution according to the high resolution mass to charge of product ions. The results show that the two compounds give stable pseudo-molecular ions (protonated molecules) in positive ion mode. Product ions for 5,6,7,4′-tetrahydroxy-flavone are mainly produced by Retro Diels-Alder (RDA) reaction from its protonated molecule M+H⁺ and subsequent neutral loss of H₂O and (or) CO, while direct neutral loss of H₂O and (or) CO from M+H⁺ give product ions with low relative abundance. Product ions for 5,6,7,4′-tetramethoxy-flavone are obtained both by direct RDA cleavage from protonated molecule M+H⁺ and neutral loss of C₂H₆ and CH₄ with subsequent RDA cleavage. More product ions for 5,6,7,4′-tetramethoxy-flavone are produced by loss of CH₄, C₂H₆, CO and H₂O, even CH₃. HR-ESI-MS/MS comparison of the two flavones shows very different MS fragmentation pathways, which leads to more special ion peaks for polymethoxy substitution than for polyhydroxy substitution.