Two approaches are proposed to measure the effect of different experimental factors (such as the modifier concentration and temperature) on the elution strength and peak shape in reversed-phase liquid chromatography, which quantify the percentage change in the retention factor and peak width (referred to the weakest conditions) per unit change in the experimental factor. The approaches were applied to the separation of a set of flavonoids with aqueous micellar mobile phases of the surfactant Brij-35 (polyoxyethylene(23)dodecanol), in comparison with acetonitrile-water mixtures, using an Eclipse XDB-C18 column. The particular interaction of each flavonoid with the oxyethylene chains of Brij-35 molecules (adsorbed on the stationary phase or forming micelles) changed the elution window, distribution of chromatographic peaks and partitioning kinetics, depending on the hydroxyl substitution in the aromatic rings of flavonoids. At 25 °C, peak shape with Brij-35 mobile phases was significantly poorer with regard to acetonitrile-water mixtures. At increasing temperature, the efficiency of Brij-35 increased, approaching at 80 °C the values obtained at equilibrium conditions, already reached with acetonitrile at 25 °C.