In order to evaluate the influence of pKa on electrophoretic mobility (EM), two novel series of synthetic flavonoids comprising eight monohydroxyflavones and four tri-O-methylluteolins were studied. In this way the effect of varying molecular size on EM was eliminated in each series. A linear relationship between EM and pKa is demonstrated for both series, with the parallel slope for the luteolins being at lower average EM due to their larger molecular size relative to the monohydroxyflavones. In an extension of these studies, the EM of a series of dihydroxyflavones was also determined. It is shown that the relative EM of a dihydroxyflavone is determined primarily by the pKa of its most acidic hydroxyl group, unless complexation with borate in the electrophoresis buffer occurs. A rationalization of the differences in EM values between the mono- and di-hydroxy series has provided an insight into the relative importance of effects such as molecular size, pKa and borate complexing on EM.