Background and purpose: The development of new drugs or formulations for central nervous system (CNS) diseases is a complex pharmacologic and pharmacokinetic process, it is important to evaluate their access to the CNS through the blood−brain barrier (BBB) and their distribution once they have acceded to the brain in order to ensure that the drug will reach its target and it will do its effect successfully. The gold standard tool for obtaining this information is the microdialysis technique, but, according to 3Rs principles it would be better having an “animal-free” alternative technique able to give the same information. Because of that, the purpose of this work was to develop a new formulation to substitute the brain homogenate in the in vitro tests used for the prediction of drugs distribution in the brain. Experimental Approach: Fresh eggs has been used to prepare an emulsion with the same proportion in proteins and lipids that a human brain. Key Results: The emulsion has proved to be able to predict, both, the unbound fraction of drug in the brain (fu,brain) and the apparent volume of distribution in the brain (Vu,brain) when tested in in vitro permeability tests. Conclusion and Implications: The new formulation could be used as a screening tool and only the drugs with a proper in vitro distribution would pass to microdialysis studies, contributing to the refinement, reduction and replacement of animals in research.