The determination of polyphenols by spectrophotometric detection is complicated due to their low concentrations in beer. The beer samples have to be pre-concentrated before using the spectrophotometric detection for their quantification. An analytical method based on solid-phase extraction (SPE) and followed by high performance liquid chromatographic separation with diode-array detection is used for the determination of free gallic, protocatechuic, caffeic, p-coumaric, ferulic and salicylic acids, of (+)-catechin, (–)-epicatechin, and quercetin. These phenolic compounds participate in colloidal and sensory stability of beer. Six different SPE cartridges were tested and three different types of elution with the most appropriate solvents (acetonitrile, acetone and methanol) were used. The performance of the HPLC method was assessed by the evaluation of parameters such as absolute recovery, relative standard deviation (RSD – lower than 10%), the limit of quantification (LOQ), and the limit of detection (LOD). The polyphenol content in various types of Czech beer is presented.