The identification and quantification of carbohydrate content in aqueous solutions and biological systems are of great interest and importance. The polar nature and large sizes of some carbohydrates can cause difficulties in identification and separation processes. Problems include long retention times and degradation during separation procedures, such as gas chromatography (GC). This project has developed methodology for an optimal quantification and identification procedure. We have derivatized carbohydrates to improve detection limits and resolution in standard GC and gas chromatography and mass spectrometry (GC/MS) analysis. This method has allowed for the identification and the separation of a synthetic mixture of 18 compounds, the qualitative identification and quantification of trace carbohydrate content in rat urine, and the analysis of cellular membrane permeability of a disaccharide. This study has the potential to make identification and quantification in biochemistry and medicinal chemistry less time-consuming and more accurate.