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A simple, precise, and rapid method was developed for the determination of four flavonoids, apigenin (AP), tricin (TR), luteolin (LU), and 4′-methoxy-tricin (MTR) in the Tibetan herbal medicine of Pyrethrum tatsienense by the high-performance liquid chromatographic technique coupled with photodiode array detection (DAD). A C18 reversed-phase column and a linear gradient elution system with acetonitrile-tetrahydrofuran-1% acetic acid as the mobile phase were employed. The method is validated in terms of the limit of detection (LOD), linearity, precision, and accuracy. The LODs were 48 ng/mL for AP, 75 ng/mL for TR, 88 ng/mL for LU, and 59 ng/mL for MTR, respectively. An excellent linear response was observed over the range specified for all analytes, as confirmed by the correlation coefficient with which from 0.9995 to 0.9999. The intraday RSDs ranged from 0.59 to 2.60%, and the interday RSDs were not higher than 0.05. The accuracy of the method was confirmed with an average recovery ranging from 95.9 to 100.0%. Since the extraction conditions can affect the extraction efficiency, nine different extracts of P. tatsienense were analyzed using different extraction solvents and extraction times. The contents of the four flavonoids in the extract of P. tatsienense were successfully determined within 18 min.