In HPLC analysis of glycoprotein glycans, oligosaccharides released from glycoproteins are often derivatized with fluorescent tags for enhancing sensitivity and selectivity. However, existing analysis methods require multiple steps for removing excess reagents and are time consuming and sometime spoil the reliability in quantitative determination. Here, we developed an online sample cleanup procedure for the analysis of 7-amino-4-methylcoumarin (AMC)-labeled oligosaccharides. An NH 2 -bonded silica packed short column (1 mm I.D. x 15 mm) and a Waters HLB TM -packed column (4 mm I.D. x 5 cm) were used for online cleanup HPLC analysis with Amide80 and ODS columns, respectively. Optimized conditions enabled the direct injection of the diluted solution of the reaction mixture onto the chromatographic system without any prior removal of the excess labeling reagents. The method was successfully applied to the analysis of N-linked glycans released from some glycoprotein samples.