No study has focused on Uvaria macrophylla leaves with various traditional efficiencies. This paper therefore applied a database-affinity ultra-high-performance liquid chromatography with quadrupole orbitrap tandem mass spectrometry (UHPLC‑Q‑Orbitrap‑MS/MS) strategy, to analyze the lyophilized aqueous extract of U. macrophylla leaves. Through database comparison and MS fragment elucidation, the study has putatively identified 41 constituents belonging to flavonoid, phenolic acid, steroid, saccharide natural product classifications. Significantly, 4 groups of isomers (liquiritigenin vs isoliquiritigenin vs pinocembrin; oroxylin A vs wogonin vs galangin 3-methyl ether; isoquercitrin vs hyperoside; protocatechuic acid vs 2,5-dihydroxybenzoic acid) have been successfully distinguished from each other. All of 41 constituents were then subjected to a quantitative analysis based on linear regression equation established by the above UHPLC‑Q‑Orbitrap‑MS/MS strategy, and an ABTS+•-scavenging antioxidant assay. Finally, the chemical content was multiplied by the corresponding ABTS+•-scavenging percentage to calculate the antioxidant contribution. It was shown that the chemical contents of 41 constituents varied from 0.003 ± 0.000 to 14.418 ± 1.041 mg/g, and gallic acid showed the highest antioxidant contribution. Gallic acid is considered as a suitable antioxidant quality-marker (Q-marker) of U. macrophylla leaves. These findings have scientific implications for resource development and quality-control of U. macrophylla leaves.