Hydrophobically modified chondroitin sulfate (CS) is widely used in the preparation of nano-sized drug delivery systems. Herein, the behavior of amphiphilic CSs in aqueous media and the drug accumulation inside the formed micelle-like structures were studied using experimental methods and molecular dynamics simulations. In particular, we focused on the impact of the degree of substitution (DS) with hydrophobic groups and the presence of drug on the morphology of the nanostructures and their molecular organization. Our results show that with increasing DS, the morphology of the amphiphilic CS nanostructures changes from irregular, loosely packed nanogels to cylindrical micelles with a core-shell architecture. These structures can efficiently accumulate hydrophobic drugs. However, the drug molecules preferentially locate at the interface between the hydrophobic part and the hydrophilic corona formed by the CS chains. Our work provides detailed information that may be relevant to the development of amphiphilic polysaccharide-based drug delivery systems.