In this research, antioxidant activities of various extracts obtained from Humulus lupulus L. were compared by DPPH, ABTS, FRAP, and CUPRAC assays. The amount of total phenolic components determined by the Folin-Ciocalteu reagent was found to be highest for 25% aqueous ethanol (9079 +/- 187.83 mg Ferulic acid equivalent/100 g extract) and methanol-1 (directly) (8343 +/- 158.39 mg Ferulic acid equivalent/100 g extract) extracts. The n-hexane extract of H. lupulus exhibited the greatest with DPPH (14.95 +/- 0.03 mu g Trolox equivalent/g sample). The highest phenolic content in the ethanolic extract could be the major contributor to its highest CUPRAC activity (3.15 +/- 0.44 mmol Trolox equivalent/g sample). Methanol-2 (n-hexane, acetone, and methanol) and methanol-3 (n-hexane, dichloromethane, ethylacetate, and methanol) extracts, respectively, exhibited the most potent ABTS (7.35 +/- 0.03 mM Trolox equivalent) and FRAP (1.56 +/- 0.35 mmol Fe2+/g sample) activities. Some of the components from the crude extracts were determined by LC-MS/MS and GC-MS analyses. Comparative screening of antioxidant activities of H. lupulus extracts and quantification of some major components by LC-MS/MS, qualitatively analysis of the reported ones which were optimal under negative ion SIM mode and coinjection, are going to be valuable for food and health applications.