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Liposome development is of great interest owing to increasing requirements for efficient drug carriers. The structural features and thermal stability of such liposomes are crucial in drug transport and delivery. Reported here are the results of the structural characterization of PEGylated liposomes via small‐ and wide‐angle X‐ray scattering and an asymmetric flow field‐flow fractionation (AF4) system coupled with differential refractive‐index detection, multi‐angle light scattering (MALS) and dynamic light scattering. This integrated analysis of the exemplar PEGylated liposome formed from hydrogenated soy phosphatidylcholine (HSPC) with the addition of cholesterol reveals an average hydrodynamic radius (Rh) of 52 nm with 10% polydispersity, a comparable radius of gyration (Rg) and a major liposome particle mass of 118 kDa. The local bilayer structure of the liposome is found to have asymmetric electronic density profiles in the inner and outer leaflets, sandwiched by two PEGylated outer layers ca 5 nm thick. Cholesterol was found to effectively intervene in lipid chain packing, resulting in the thickening of the liposome bilayer, an increase in the area per lipid and an increase in liposome size, especially in the fluid phase of the liposome. These cholesterol effects show signs of saturation at cholesterol concentrations above ca 1:5 cholesterol:lipid molar ratio. [ABSTRACT FROM AUTHOR]