Technical Abstract: In natural product chemistry, it is often crucial to determine sugar composition as well as the absolute configuration of each monosaccharide in glycosides. An ultra-performance liquid chromatography method using both photodiode array (PDA) and mass spectrometry detectors (UPLC-UV/MS) was developed. Within a single injection, 16 monosaccharide derivatives including 6 pairs of aldose enantiomers and 4 other monosaccharides were identified in less than 25 minutes. It was determined that the structures of derivatives of sugar enantiomers correlated with retention time. The UPLC-MS method can increase sensitivity of detection of the saccharides by more than 10 times compared to previously reported methods. The one-pot reaction of monosaccharide with L-cysteine methyl ester and phenyl isothiocyanate is easily reproduced, clean and relatively simple in a screw-capped reaction vial. The developed method was successfully applied for the analysis of different of glycosides, viz., glycosides of triterpenes, steroids and flavonoid which commonly exist in nature. The absolute configurations of composed monosaccharides are clearly characterized from tested samples.