High-resolution ultrasound spectroscopy (HR-US), size and ζ-potential titrations, and isothermal titration calorimetry (ITC) were used to characterize the interactions between hyaluronan and catanionic ion pair amphiphile vesicles composed of hexadecyltrimethylammonium-dodecylsulphate (HTMA-DS), dioctadecyldimethylammonium chloride (DODAC), and cholesterol. In addition to these methods, visual observations were performed with the selected molecular weight of hyaluronan. A very good correlation was obtained between data from size titration, HR-US, and visual observation, which indicated in lower charge ratios the formation of hyaluronan-coated vesicles. On the contrary, at higher charge ratios, coated vesicles disintegrated to a size of around 2000 nm. The intensity of these interactions and the disaggregation were dependent on the molecular weight of hyaluronan. All interactions studied by ITC showed strong exothermic behavior, and these interactions between vesicles and hyaluronan were confirmed from the first addition, independently of the molecular weight of hyaluronan.