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Bioactive compounds extracted from plant essences are of great value for those enterprises interested in natural products; plant tissue culture techniques guarantee a reliable and constant biomass production. Hop (Humulus lupulus L.), with its wealth in bioactive compounds, is an unvaluable resource. The present study focused on the characterization of vitro-derived leaves and roots of two hop genotypes, Cascade and Gianni. Extracts obtained from the selected hop explants were investigated, determining their polyphenolic content as their antioxidant capacity, applying DPPH, ABTS and FRAP assays; moreover, phenolic extract molecular profile was obtained through UHPLC-MS/MS. Results confirm the wealthy in bioactive compounds and the antioxidant properties of vitro-derived hop explants tested; furthermore, the qualitative characterization evidenced that extracts obtained from vitro-derived hop tissues are composed of twenty one different compounds and contain the same hop antioxidant molecules, as well as xanthohumol and isoxanthohumol, also present in open field grown hop plants, such as α-acids and β-acids. Achievement obtained are encouraging and lays the groundwork to further investigate the potential of vitro-derived hop plantlets as bioactive compounds source.