Hops are used almost exclusively for bitterness and flavor by brewers. We propose the first analytical application of CZE coupled to ESI-MS for the separation and structural elucidation of organic compounds in the methanolic extracts of hops, and different extraction procedures of the plant material have been carried out. The proposed method permits the identification of hop polyphenols (flavonoids glycosides and chalcones), bitter acids (alpha-acids and beta-acids), and their oxidation products. The optimization of CZE parameters (pH, concentration, and type of buffer) and ESI-MS parameters (nature and flow rate of the sheath liquid, nebulizer pressure, drying gas flow rate, temperature, and compound stability) have permitted the development of a rapid, simple, direct, and straightforward CZE-ESI-MS method for the identification of components of methanolic extracts from different hops used in the brewing process.