Objective To develop a method for determination of positional isomer ratio of PEGylated recombinant human interferon(PEG-rhIFN)α2 b by ion exchange high performance liquid chromatog.(IEC-HPLC), and analyze the modification sites of various positional isomers. Methods HPLC anal. was performed on a Proteomix SCX-NP10(10. 0 mm x250 mm, 10 μm) column. The mobile phase A was 1. 8 mmol/L citric acid monohydrate-3. 3 mmol/L disodium phosphate dodecahydrate(pH 5. 3), while the mobile phase B was 30 mmol/L trisodium citrate dihydrate-35 mmol/L sodium dihydrogen phosphate dehydrate(pH 6. 0). Linear gradient elution(0 ∼ 180 min:0% B → 12% B;180 ∼ 220 min:12% B → 15% B)was adopted at a flow rate of 0. 4 mL/min, a column temperature of 25 °C and a sample load of not less than 100 μg. The IEC-HPLC method was verified for specificity and reproducibility. The positional isomer ratio in three batches of PEG-rhIFNα2 b was determined by the developed method, and the result was analyzed by using SPSS 19. 0 software to propose the quality standard for ratios of five positional isomers. The elution peaks of five positional isomers were enriched by the developed method to fix the positions of modification sites. Results No chromatog. peak of blank control or rhIFNα2 b appeared, and there was no interference to the determination of positional isomer in PEGrhIFNα2 b. Five peaks of the isomers with good separation degrees were observed The RSDs of areas of peaks 1, 2, 3, 4 and 5 in the same batch of PEG-rhIFNα2 b determined for 3 consecutive day were 1. 96%, 2. 53%, 2. 54%, 1. 05%and 1. 86% resp., all of which were less than 3%. The quality standards for the ratios of five positional isomers in PEG-rhIFNα2 b were proposed as 5% ∼ 9%(peak 1), 14% ∼ 21%(peak 2), 7% ∼ 10%(peak 3), 30% ∼ 34%(peak 4)and 31% ∼ 39%(peak 5)resp. The modification sites of positional isomers were mainly K31 for peak 1, K134 for peak 2, K131 and K164 for peak 3, K83 for peak 4, and K121 for peak 5. However, no PEG-modified N-terminus was detected. Conclusion An IEC-HPLC method for determniation of positional isomer ratio of PEG-rhIFNα2 b was developed, which showed high specificity and reproducibility.