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The use of guayusa (Ilex guayusa Loes.) leaves as functional food has increase recently. This work discusses the antioxidant activity and volatile compounds of guayusa leaves extract and fractions. The methanol crude extract was obtained by maceration, subsequently hexane, chloroform, ethyl acetate, and aqueous fractions were collected by solvent-solvent partition.Total phenolic content (TPC), total flavonol/flavone content (TFC), 2,2-diphenyl-1picrylhydrazyl (DPPH) radical scavenging activity, and ferric reducing antioxidant power (FRAP) were measured by ultraviolet-visible (UV-Vis) spectrophotometry. The results revealed that ethyl acetate fraction showed highest inhibition against DPPH radical (93.86±0.95%) at 500 µg/mL, and reduce the ferric-tripyridyltriazine complex (Fe 3+ -TPTZ) at 1619.81 mg trolox equivalent (TE)/g, followed by aqueous fraction. This bioactivity could be related to phenolic acids, flavones and flavonols content, as well as the caffeine, dodecanoic acid isopropyl ester, caffeic acid, and malic acid identified by gas chromatography-mass spectrometry (GC-MS). These findings support the antioxidant properties of this plant material.