Hydroxyethyl starch (HES) is a water soluble semisynthetic polysaccharide that is used as a plasma volume expander and cryoprotectant. In order to produce a fully biodegradable amphiphilic polymer, HES was esterified with lauric, palmitic, and stearic acids under mild reaction conditions using dicyclohexyl carbodiimide (DCC) and dimethylaminopyridine (DMAP). The molar substitution of the acyl chains (MSfatty acid) was determined with H-1 NMR spectroscopy, while the conformational state of the hydrocarbon chains in the graft copolymer was determined using Raman spectroscopy. Furthermore, the aqueous self-assembly of the modified polymer was studied using dynamic light scattering (DLS) and transmission electron microscopy (TEM). Results show the formation of 20 to 30 nm micelles, and 250 to 350 nm polymeric vesicles. Electron spin resonance (ESR) spectroscopy was used to study the microenvironment of a hydrophobic spin probe loaded inside the formed nanodispersion. It was possible to identify the location of the probe and its distribution between the micelles and vesicles. Finally, the hydrophobically modified HES might find use as a potential drug carrier, warranting the future investigation of its ability to encapsulate and deliver drug candidates.