RP-HPLC method with a simple “MeOH-H₂O-TFA” elution system is applied in purification and analysis of human glycoalbumin fragment including the region of lysine 525, which was synthesized by different methods. The peptide fragment of albumin KQTALYYC prepared by acid-labile PEG resin was glycosylated and purified by gel filtration and analysed by RP-HPLC. A single peak demonstrated that the glycosylation of the peptide was successful. Heptapeptide fragment of albumin QTALYYC was synthesized by chloromethyl resin according to the Boc chemistry strategy. The pure heptapeptide was obtained by semi-preparative RP-HPLC. Glycosylated lysine was purified by gel filtration and ascertained by RP-HPLC. The heptapeptide was coupled with glycosylated lysine. RP-HPLC of the product showed that the glycopeptide is qualified as a semiantigen.