Amino sugars have been used as biomarkers to assess the relative contribution of dead microbial biomass of different functional groups of microorganisms to soil carbon pools. However, little is known about the dynamics of these compounds in soil. The isotopic composition of individual amino sugars can be used as a tool to determine the turnover of these compounds. Methods to determine the δ13C of amino sugars using gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) have been proposed in literature. However, due to derivatization, the uncertainty on the obtained δ13C is too high to be used for natural abundance studies. Therefore, a new high-performance liquid chromatography/isotope ratio mass spectrometry (HPLC/IRMS) methodology, with increased accuracy and precision, has been developed. The repeatability on the obtained δ13C values when pure amino sugars were analyzed were not significantly concentration-dependent as long as the injected amount was higher than 1.5 nmol. The δ13C value of the same amino sugar spiked to a soil deviated by only 0.3‰ from the theoretical value. Copyright © 2009 John Wiley & Sons, Ltd.