A new quantification method for hop-derived bitter compounds in beer was developed. By means of LC-MS/MS operating in the multiple reaction monitoring mode, a total of 26 hop-derived bitter compounds, namely, the post-, co-, n-, ad-, pre-, and adpre-congeners of iso-alpha-acids, alpha-acids, and beta-acids, as well as the prenylflavonoid isoxanthohumol and the chalcone xanthohumol, could be simultaneously detected for the first time in a single HPLC run in authentic beer samples without any cleanup procedures. To compensate for the effect of coextracted matrix components in LC-MS/MS analysis, the so-called ECHO technique was applied for the first time as a suitable strategy for the quantitative analysis of the hop-derived bitter compounds in fresh and stored beer. On the basis of quantitative data, the remarkable instability of alpha-acids and trans-iso-alpha-acids was confirmed, and it was observed that the degradation of trans-iso-alpha-acids during the storage of beer is not dependent from the nature of the alkanoyl side chain of the congeners. In contrast, an increase of the concentrations of beta-acids and of the prenylflavonoid isoxanthohumol as well as of the chalcone xanthohumol during the storage of beer was observed.