Altered glycan levels in serum have been associated with increased risk of cancer. In this study, we have developed and validated a HPLC-based method to analyze monosaccharide composition (D-mannose, Glucosamine, Galactosamine, Glucuronic acid, D-glucose, D-galactose, D-xylose, L-fucose) in human serum, with L-rhamnose, being used as internal standard. Monosaccharides obtained from hydrolyzed serum samples were derivatized by 1-Phenyl-3-methyl-5-pyrazolone. A ZORBAX XDB-C18 column(150x4.6mm) was used for chromatographic separation with 100 mM ammonium acetate buffer (NH4Ac-HAc, PH=5.5, solvent A), acetonitrile (ACN, solvent B) as a mobile phase. The calibration standard curves for the eight monosaccharides showed good linearity over the range of 2.5-500 mu g/mL with R-2 > 0.995. The relative standard deviation values for intra-day and inter-day precision were <= 5.49%. Recovery was 69.01-108.96%. We observed that this column exhibited high specificity and selectivity to separate monosaccharides from serum. This method was then applied to quantitatively analyze the serum monosaccharide levels in 30 patients with endometrial cancer and 30 matched healthy controls. Statistical analysis indicated that the serum monosaccharide levels were significantly higher in patients compared with healthy controls (P value< 0.0001). Overall, we report here a simple, reliable, low-cost, and reproducible HPLC method for the separation and quantification monosaccharides in the human serum, which has potential value to serve as a screening marker for endometrial cancer.