Menopause is a time in a women`s life when her periods eventually stop and the body goes through many changes accompanied by numerous unpleasant menopausal symptoms. Today, women enter the menopause earlier than couple of decades ago. In many women, symptoms of menopause are so strong that they interfere with their daily life activities. For that reason, many women use dietary supplements for the relief of these uncomfortable symptoms. More and more dietary supplements for menopausal symptoms appear on the market and therefore there is a growing need for quality assessment of these products. Development of new, selective, sensitive and accurate analytical methods for the analysis of active compounds in such products is very important. The aim of this study was to optimize extraction conditions for the analysis of active phytoestrogenic ingridients and their quantitative determination in various herbal material and dietary supplements. For the optimization of extraction conditions herbal material of 3 species (Glycine max L., Vitex agnus castus L., Humulus lupulus L.) was used. Material was homogenized and the active ingredients were extracted by ethanol and acetonitrile. Extraction solvents used were of 100%, 75% and 50% concentractions. Also, different extraction time, 15 min, 30 min and 60 min, was investigated. Extraction was performed at room temperature using an ultrasonic bath. After the analysis of the samples by newly developed and validated LC/DAD/MS/MS method, using Agilent 1100 Series LC/MSD trap system, best extraction solvent for flavonoids in Humulus lupulus proved to be 100% ethanol at 15 minutes extraction time.