The main flavonoid glycosides from Betulae folium are separated with a RP-HPLC method. The mobile phase for the separation of the seven most important compounds (rutin, hyperin, quercetin-3-arabinofuranoside, quercetin-3-arabinopyranoside, quercetin-3-rhamnoside, quercetin-3-glucuronide, and myricetin-3-galactoside) was optimized with the “PRISMA” model, which is a multisolvent optimization system. The isocratic separations were finished within 30 minutes and the peak purity was controlled with diode-array detection. The method can be applied for quantitative determination, as was demonstrated for different samples of B. pendula and B. pubescens.