Separation of glucose from mixtures of fructose and sucrose in molasses is a major challenge in industrial sugar chromatographic separations. The efficiency of a chromatographic process is largely dependent on the adsorbent used. Sulfonated poly(styrene-co-divinylbenzene) (PS-DVB) ion exchange resins are the most frequently used for sugars separation, generally in a cationic form. The cation will complex with the hydroxyl group of the sugar leading to a selective adsorption according to the orientation of the hydroxyl group. Thus, the conformation of the sugar determines its relative affinity for the resin and its distribution coefficient. Consequently, fructose forms the more stable sugar-cation complex being preferentially adsorbed in the resin followed by glucose. Sucrose interacts very weakly with the cation and is partially or totally excluded from the resin matrix owing to its larger molecular size. The separation process is usually carried out at high temperatures. However, this implies high energy costs and an elevated level of hydrolysis. Adsorption isotherms of the sugars present in a mixture are a very important parameter for the selection of the adsorbent to be used in the chromatographic separation. Therefore, in this study, the adsorption isotherms of glucose, fructose and sucrose were determined for two resins of PS-DVB in the sodium and potassium forms. Potassium and sodium are the most recommended cations for the separation of sucrose, fructose and glucose. The sodium and potassium resins matrix used in this work consist in meshes of 500-600 μm and 290-350 μm, respectively. Solutions with single and multi-component mixtures of the three sugars were used at 25ºC and 40ºC. A static method was used to determine the equilibrium adsorptions: 3.5 mL of solutions with different sugar concentrations were added to a known amount of adsorvent. The mixture was held for 8h under agitation and a fixed temperature and a final concentration in equilibrium with resin was determined by HPLC.