A rare sugar is considered to be a monosaccharide rarely found in nature. To investigate their natural distribution and biological roles, a robust analytical system must be used to isolate, identify, and quantify them. Herein, we report the development of such a system that can specifically quantify and chromatographically separate four aldopentoses and eight aldohexoses tagged with 2-aminopyridine. Purified monosaccharides derivatized with a pyridylamino moiety (PA-monosaccharides) are first chromatographed over a high-performance anion-exchange resin. But, because two of the PA-aldohexoses used in this study, PA-talose and PA-idose, co-elute with the common saccharides, PA-glucose and PA-mannose, respectively, a second chromatographic step, reversed-phase high-performance liquid chromatography, is used to completely separate them. Thus, as shown by the results of this study, chromatographic separation of PA-monosaccharides is achievable and provides a quantitative measurement of common and rare isomeric aldopentoses and aldohexoses.