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Guayusa tea is derived from the leaves of the Ilex guayusa Loes. plant, which is native to the Amazon rainforest. Beyond its pleasant sensory properties, Guayusa tea is rich in antioxidants, phenolics, and minerals. In this study, the effects of infusion time, temperature, and solvent conditions on the color, antioxidant capacity, total phenolic content, phenolic profile, and antimicrobial activity of Guayusa (Ilex guayusa Loes.) tea were investigated. Guayusa tea samples were prepared using two different solvents, ethanol and water, with 4, 6, and 8-h infusions at 60 and 70 °C. Liquid chromatography, diode array detection, and electrospray ionization tandem mass spectrometry (LC-DAD-ESI-MS/MS) were used to determine a comprehensive profile of phenolic compounds and to detect differences due to infusion conditions. Moreover, after the Guayusa tea infusion with the highest bioactive properties was determined, the effects of in vitro gastrointestinal digestion on the total phenolic content, antioxidant capacity, and phenolic compounds of the Guayusa tea infusion were measured. Phenolic profile analysis identified 29 compounds, among which chlorogenic acid and its derivatives were predominant. The increase in infusion time was correlated with an elevation in total phenolic content. Significant differences were observed between water and ethanol infusions of Guayusa in terms of phenolics and antioxidants. The total amount of phenolic compounds in the samples prepared with both solvents was found to increase after oral intake, depending on the digestion stage; meanwhile, the amounts of flavonoid compounds and di-O-caffeoylquinic acid derivatives decreased during digestion. [ABSTRACT FROM AUTHOR]