Insulin was modified with monomethoxypolyethylene glycol (MPEG)-succinimidyl succinate and succinimidyl ester of carboxymethyl MPEG. Effects of reaction solvents, initial molar ratio of MPEG derivative to insulin and reaction time on PEGylation of insulin were investigated by 2,4,6-trinitrobenzenesulfonic acid spectrophotometric assay and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sephadex G75 size exclusion chromatography (SEC), ion exchange chromatography (IEC) and reversed phase-high performance liquid chromatography (RP-HPLC) were applied to separate PEGylated insulin. IEC and RP-HPLC were proved to be efficient tools on separation of different PEGylated insulin species.