Objective To develop and verify a reverse-phase high performance liquid chromatog.(RP-HPLC) for determination of Me p-hydroxybenzoate and Pr p-hydroxybenzoate contents in human interferon(IFN) α2a injection. Methods C_(18) column(5 μm, 250 mm x 4. 6 mm)was adopted, serving methanol-1% glacial acetic acid(60 :40)as mobile phase at a flow rate of 1 mL/min, a detection wavelength of 254 nm, and a column temperature of 25 °C. The result was calculated by standard curve method. The developed method was verified for specificity, linearity, accuracy, precision and durability, by which the Me p-hydroxybenzoate and Pr p-hydroxybenzoate contents in six batches of IFNα2a injection were determined Results All the resolutions between chromatog. peaks of the components to be determined and between the chromatog. peaks of the components to be determined and other chromatog. peaks were more than 1. 5, indicating a good specificity of the method. The linear ranges of Me phydroxybenzoate and Pr p-hydroxybenzoate contents were 0. 002% ∼ 0. 012% and 0. 000 2% ∼ 0. 001 2% resp. The average spike recoveries of Me p-hydroxybenzoate and Pr p-hydroxybenzoate were 99. 6% and 99. 2%, resp. The RSDs in reproducibility test for Me p-hydroxybenzoate and Pr p-hydroxybenzoate were 0. 05% and0. 07% resp. The method showed good durability for different brands of chromatog. columns, different proportions of mobile phase components and different column temperatures The contents of Me hydroxybenzoate and Pr p-hydroxybenzoate in six batches of samples were 0. 058% ∼ 0. 060% and 0. 005 7% ∼ 0. 005 9% resp., which were significantly closed to the feeding amounts Conclusion The developed RP-HPLC method is simple, specific, linear, accurate, precise and durable, which may be used for the determination of Me p-hydroxybenzoate and Pr phydroxybenzoate contents in human IFNα2a injection instead of gas chromatog.