In the paper, the identification and quantification of α- and β-amyrin in five varieties of Humulus lupulus is described. The plant samples were extracted with ethyl acetate and analyzed using of high performance liquid chromatography (HPLC) with PAD detection. The separation was achieved on RP 18 column, at 2 mL/min flow rate and at temperature of 35°C. Acetonitrile was used as a mobile phase. The established calibration curves and the other validation parameters: linearity (correlation coefficient r > 0.9988) and precision (RSD values ranged from 0.14 to 1.81%) were found to be satisfactory for the proposed method. The content of α- and β-amyrin strongly depended on varieties of H. lupulus and amounted from 72.7 to 232.5 μg/g and from 77.9 to 176.9 μg/g of dry plant material, respectively.