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Information on the chemical composition of Osmanthus fragrans fruits is still limited because there are many compounds present in low concentrations in the plant. In this work, the bioactive components in O. fragrans fruit extract were investigated by a new high-performance liquid chromatography electrospray ionization tandem mass spectrometry method, which allows sensitive analysis both in identification and quantitation. A total of 28 compounds were tentatively identified, and 16 components were discovered in O. fragrans fruits for the first time. The validated quantitative methods for the determination of the bioactive components were subsequently applied to analyze batches of O. fragrans fruits from different cultivars, which is beneficial for the comprehensive utilization of O. fragrans fruits.