A set of biorelevant media "fasted-state simulated intestinal fluid with cholesterol (FaSSIF-C)" for the in vitro study of intestinal drug dissolution in the duodenum was developed. These contain cholesterol at the same levels as in human bile: the cholesterol content of FaSSIF-7C is equivalent to healthy female, FaSSIF-10C to healthy male persons, and FaSSIF-13C to several disease cases that lead to gallstones. The fluids were studied in three aspects: biocompatibility, intestinal nanostructure, and solubilizing power of hydrophobic drugs of the BCS class II. The biocompatibility study showed no toxic effects in a Caco-2 cell system. The drug-solubilizing capacity toward Fenofibrate, Danazol, Griseofulvin, and Carbamazepine was assessed as example. It varied with the cholesterol content widely from a fourfold improvement to a twofold reduction. The nanostructure study by dynamic light scattering and small-angle neutron scattering indicated vesicles as the main component of FaSSIF-C in equilibrium (>1 h), but at high cholesterol content, larger particles were observed as a minor contribution. The neutron experiments indicated the presence of complex micelle-vesicle mixtures, even after 1 h development of fed-state bile model to FaSSIF. The results indicate that cholesterol affects some drugs in solubilization and particle size in intestinal model fluids.