An empirical method was developed to determine the absolute configuration of monosaccharides, based on high-field 1 H NMR spectroscopy of their per-O-(S)-2-methylbutyrate (SMB) derivatives. The SMB derivatives of the d and l forms of a given monosaccharide are diastereomers, allowing them to be distinguished on the basis of differences in their 1 H NMR chemical shifts. The reproducibility of these chemical shift differences may allow the absolute configuration of a wide range of monosaccharides to be routinely determined by comparing the spectra of their SMB derivatives to standard spectra in a data base, making it unnecessary to prepare and analyze new standards for each analysis. The derivatization procedure uses inexpensive, easy-to-handle reagents and is virtually complete. Application of the method to three complex glycans provided an unambiguous determination of the absolute configurations of their constituent monosaccharides.