The measurement of intestinal permeability is important for diagnoses of diseases of the gastrointestinal tract, such as Crohn’s disease. The gold standard for measurement of intestinal permeability is the dual sugar absorption test, which measures the urinary or blood concentrations of two orally administered sugars, a monosaccharide and a disaccharide, over a period of time. The lining of the small intestine allows monosaccharides to cross into the bloodstream, but the larger disaccharide is not permitted to cross the intestine unless the barrier is compromised. The permeability of the lining is measured by a ratio between select monosaccharides and disaccharides, and this indicates the overall status of the small intestine. In order to study the effects of resistance exercise on intestinal permeability in human subjects, we developed a liquid chromatography tandem mass spectrometry (LC–MS/MS) method for the analysis of saccharides in blood plasma. The analytes included rhamnose, a monosaccharide not commonly found in food, and lactulose, a disaccharide. A trisaccharide, raffinose, was used as an internal standard. The method was robust, and had consistent reliability.