This work is aimed at developing a new process to separate alpha-cyclodextrin (CD) from binary alpha-CD/beta-CD mixtures. To achieve this objective, a membrane process was designed with water as solvent and using the selective accommodation of a guest Igepal Co-890 (IG) within the beta-CD cavity. In the first part, host-guest inclusion study suggested the formation of IG(2)/beta-CD complex in presence of IG excess. Moreover, increasing IG/CD molar ratio to 1.5 leads to the complete complexation of beta-CD while alpha-CD remains free. The second part was devoted to the separation of alpha- and beta-CD using UF membranes (GH and GK from GE-Osmonics). The influence of parameters including membrane molecular weight cut-off (MWCO), transmembrane pressure (Delta P), IG/beta-CD and alpha-CD/beta-CD molar ratios, was investigated. MWCO, Delta P, IG/beta-CD and alpha-CD/beta-CD molar ratios respectively of 1000 Da, 2 bar, 1.5 and 11:89 are the optimized values to reach an alpha-CD separation factor as high as 38. The experimental data fits very well with predicted values calculated using the parameters found in the complexation study and rejection of single solutions indicating that selectivity predominantly originates from steric exclusion of species by membrane GH.