Hydrophobic interaction chromatography (HIC) is presented as a suitable method to determine the surface hydrophobicity of colloidal drug carriers. The experimental design is described in great detail. Results obtained with surface-modified polystyrene particles as model carriers and parenteral fat emulsions are discussed as examples. HIC was able to distinguish between particles chemically modified by the introduction of functional groups. Polystyrene particles of various size were surface-modified by adsorption of the block-polymer Poloxamine 908. HIC distinguished between the hydrophobicities of these adsorption layers. The measured surface hydrophobicities of particles and fat emulsions were well in agreement with the in vivo organ distribution data obtained after i. v. injection of the carriers.