Abstract: OBJECTIVE To establish a method for simultaneous determination of naringin, hesperidin and neohesperid in Citrus changshan-huyou fruit in different growth periods by HPLC, to study the dynamic changes of three contents, and then to determine the best harvest period of Citrus changshan-huyou as the medicinal resource. METHODS The analysis was performed on an Agilent ZORBAX SB-C₁₈ column (4.6 mm×250 mm, 5 μm) with acetonitrile-0.2% aqueous phosphoric acid (15∶85) as the mobile phase at the flow rate of 1.0 mL·min^-1. The detection wavelength was set at 284 nm. And column temperature was 40 ℃. The analysts were quantified by external standard method using peak areas. RESULTS The linear ranges were 28.45-284.50 ng(r=0.999 8) for naringin, 18.09-180.93 ng(r=0.999 8) for hesperidin, and 85.86-858.55 ng(r=0.999 6) for neohesperidin. The average recoveries were 98.83%(RSD=2.12%) for naringin, 99.05%(RSD=2.30%) for hesperidin and 98.83%(RSD=1.85%) for neohesperidin. The contents of naringin, hesperidin and neohesperidin in Citrus changshan-huyou fruit in different growth periods were determined simultaneously. As a result, the content ranges were 12.65-109.66 mg·g^-1 for naringin, 0.45-17.50 mg·g^-1 for hesperidin, and 8.06-218.90 mg·g^-1 for neohesperidin, respectively. Naringin had the highest content in June, the highest in May for hesperidin and neohesperidin. The contents of naringin and neohesperidin in Citrus changshan-huyou harvested during May to July met the requirements of Chinese pharmacopoeia. CONCLUSION The described HPLC method is simple, accurate and reliable, and is suitable for the determination of naringin, hesperidin and neohesperidin. The results provide reference for determining the best harvest period of Citrus changshan-huyou as the medicinal resource.