A sensitive method for determining the total amount of sialic acid, glucosamine, and galactosamine in blood serum and glycoconjugates using reversed phase high performance liquid chromatography is described. Sialic acids present in glycoconjugates are released by acidic hydrolysis with 2 M TFA at 80°C for 2 h, whereas hexosamines are released following hydrolysis with 6 M HCl at 100°C for 8 h. The sialic acids released under these conditions are identified as a decarboxylated and deacylated product bearing a primary amino group (amino-product). Following removal of neutral monosaccharides and amino acids by ion-exchange chromatography on Dowex 50x8, the obtained amino-product and hexosamines are converted to Tosyl-derivatives. The obtained derivatives of amino-product, glucosamine, and galactosamine, are completely separated and determined on a Supelcosil LC-18 column by means of isocratic elution using a mobile phase of water-acetonitrile (85:15, v/v) and detection at 231 nm. The method shows a linearity range up to 25 nmol of sialic acids and hexosamines, has a detection limit ranging from 6 to 12 pmol, and is highly accurate when compared with literature values. The present method has the advantage of permitting determination of total sialic acid, glucosamine, and galactosamine in glycoconjugates and blood serum using the same HPLC conditions.