An optimization procedure for the separation of fJavonoids from St John's Wort by reversed- phase high-performance liquid chromatography on monolithic stationary phase is described. Three-component mobile phase systems are studied using experimental design methodology The starting experimental domain is a triangle, each vertexofwhich is a pure component. From preliminary isocratic experiments, truncations in the domain are performed leading to a quadrilateral shape working domain with no symmetry To cope with both separation and analysis time, desirability functions are used. Optimal conditions are finally given by binary systems and the four flavonoids are separated in less than seven minutes.