A new HPLC method with coulometric detection for the quantification of xanthohumol, alpha-acids and beta-acids in hops was developed. The separation of compounds was accomplished with a C18 column and isocratic elution with methanol: 50 mM potassium phosphate: ortho-phosphoric acid=80:20:0.25 (v/v/v). The method was validated and UV and electrochemical detectors (ECD) were compared. The HPLC method with ECD was precise, accurate and very sensitive for detection of xanthohumol and alpha- and beta-acids. The detection limits of analytes were at least 8.8 to 24 times lower with ECD than those of the UV detector. The ECD method was successfully applied for quantification of studied compounds in hop pellets. The concentrations of all compounds obtained with ECD and UV were found to be equivalent. This is the first study demonstrating a very sensitive and validated method for the quantification of xanthohumol, alpha- or beta-acids in hop samples with the use of the electrochemical detector.