A series of N,O-acylated chitosan derivatives were emulsified with different fatty acids. Hydrophobically modified chitosan derivatives were expected to exhibit self-assembly behaviour resulting in micelle formation. Several parameters of the oil-in-water emulsification process were investigated: mixing method, speed and duration, volume oil phase and addition of modifiers. Their influence on micellar Z-average diameter, size distribution and Zeta potential was analysed based on dynamic light scattering measurements. There were various relations between the hydrodynamic behaviour of chitosan derivatives, their chemical structure and the process parameters. Additionally, the obtained micelles could serve as active compound carriers because they encapsulated two model substances, namely ibuprofen and α-tocopherol.