From a molecular point of view, transport of small molecules across lipid bilayers is a fundamental and functional process. The release of efficacious dose of bioactive-entrapped in liposome depends on different parameters such as liposome permeability, bioactive structural properties and strength of liposome / bioactive interaction. The aim of this study was investigation the possible mechanisms of hydrophilic molecules transfer through liposomal bilayer. Calcein was chosen as model of hydrophilic drugs. In the first step, we optimized liposome formulation by considering its physicochemical properties (size, encapsulation efficiency, fluidity and etc.) by different methods such as DSC, TEM, SAXS, DLS, NMR and Spectroufluremtere. The reported results show that mean size, zeta potential, Tc, entrapment efficiency and fluidity were influenced by liposome lipid composition. Then, we tried to investigate hydrophilic bioactive agents? interaction with liposome by Raman Spectroscopy, Langmuir Balance and Differential Scanning Calorimetry. The obtained results indicated that calcein is being able to interact with the choline polar-head group of the lipids but probability it could intercalate into the acyl chains and disturb the chain order. Finally, the permeability of calcein across some liposome membranes was first evaluated on the basis of the first-order kinetics by spectrofluorometer. Second, the composition/fluidity effect of liposome as well as the incubation temperature/pH effect was investigated. Furthermore, a model simulating the conditions of digestion was developed to estimate the partition coefficient and to determine the mechanism transfer through liposomal bilayer by using AFM and STED methods. The results confirmed that calcein permeates slowly through liposomal membrane by diffusion without liposome disruption