In this study the possibility of derivatizing sugars using microwave irradiation was investigated. The amount of reagent, irradiation intensity, and derivatization time were optimized. In the derivatization of sugars with p-nitroaniline the reaction is complete within 5 min at 600 W when the p-nitroaniline-to-sugar and NaBH3CN-to-sugar mole ratios were above 1.4 and 3.1, respectively. A Doehlert design was used to optimize the mobile phase for separation of p-nitroaniline-labeled sugars; and the best separation was obtained by use of 0.01 mol L-1 acetate buffer at pH 4.40 containing 11.0% acetonitrile. Analysis using this method was highly sensitive and analysis time was short. Finally, a food sample was analyzed using the proposed method.