A [superscript 1]H NMR spectroscopy-based laboratory experiment explores mono- and disaccharide structural chemistry, and the enzyme-substrate specificity of glycosidic bond cleavage by ß-glucosidase towards cellobiose (ß-linked gluco-disaccharide) and maltose (a-linked gluco-disaccharide). Structural differences between cellobiose, maltose, and the glycolytic product glucose are established in terms of chemical shift changes and J-coupling constants of the anomeric proton peaks. The distinct anomeric proton signals observed in the 4.0-5.5 ppm range of glucose, cellobiose, and maltose spectra serve as a signature NMR feature to monitor the substrate specificity of ß-glucosidase towards cellobiose as a substrate. This experiment consolidates carbohydrate structural chemistry and NMR spectroscopy as applied to investigate enzyme specificity in the context of a biochemistry laboratory experiment with hands-on NMR experience for upper-division undergraduates.