Abstract: An HPLC method was established to the determination of five flavonoids (vicenin-2, lonicerin, rhoifolin, luteolin and apigenin) in Pteris multifida. Separation was carried out on a Diamonsil C18 column (4.6 mm×250 mm, 5 μm). The mobile phase was acetonitrile-water (containing 0.1% formic acid) with gradient elution (0-10 min, 15:85; 20-30 min, 25:75; 35-40 min, 50:50). The flow rate was 1.0 mL/min at 25 ℃, and the detection wavelength was set at 350 nm. The average recoveries for vicenin-2, lonicerin, rhoifolin, luteolin and apigenin were 97.18%-105.20% (RSD1.72%-3.20%), 95.02%-104.92% (RSD 1.96%-2.93%), 98.45%-103.70% (RSD 1.66%-2.70%), 96.41%-104.73% (RSD 1.03%-3.46%), 95.82%-99.34% (RSD 1.85%-3.95%), respectively. These compounds showed good linearity(r≥0.999, n=5) in the detected concentration ranges. There were no significant differences of mass factions of the five flavonoids from different regions. The total mass factions of the five flavonoids were in the range of 0.5-2.2 mg/g.