The fruit bodies of Fomes officinalis are used for the treatment of coughs, gastric cancer, rheumatism and hydropsia; however, no method is currently available to assess the quality of this medicinal fungus based on quantitative profile of its main triterpenes. To develop a simple and accurate HPLC-UV method for the simultaneous quantification of five lanostane-type triterpenes in the fruit bodies of F. officinalis. Separations were performed on an Agilent Zorbax Eclipse XDB-C(18) column by gradient elution using acetonitrile : formic acid. Analytes were identified by HPLC coupled with electrospray ionisation mass spectrometry experiments. The quantitative HPLC-UV method was validated for linearity, precision, accuracy and limits of detection and quantification. Calibration curves presented good linear regression (r > 0.9996) within test ranges. The relative standard deviation of this method was less than 1.7% for intra- and inter-day assays and overall recoveries were 96.4-104.1% for the five compounds analysed. The method was successfully applied to the quantification of five triterpenes in 16 samples of F. officinalis collected from different regions. The developed assay could be considered as a suitable quality control method for F. officinalis.