Hop (Humulus lupulus L.) is grown mainly for the production of beer. The flowers of the female plant give it the bitter taste and pungent aroma. There are a large number of hop varieties differing in their α-acid content, essential oil levels and odor profiles. Aside from their use in brewing, more recently, hops have been used for the pharmacological properties of its derivatives that are of great importance to the pharmaceutical industry. Hop is known to have a fairly complex chemistry characterized by the presence of a variety of sesquiterpenoids, diterpenoids and triterpenoids, phytoestrogens and flavonoids. Additionally, considering the countless applications in the pharmacological sector in recent years, a chemical characterization of the different cultivars is essential to better identify the source of specific secondary metabolites. For this purpose, the dried inflorescences of two hop cultivars, Chinook and Cascade, were investigated using Solid-Phase Microextraction-Gas Chromatography-Mass Spectrometry and Liquid Chromatography-Tandem Mass Spectrometry (SPME-GC-MS and LC-MS-MS) to describe their metabolomic and proteomic profile. Furthermore, thanks to an in-depth statistical survey, it was possible to carry out a comparative study highlighting interesting implications deriving from this investigative study.