In CNS drug discovery, knowledge of drug-tissue binding is essential for a better understanding of brain penetration by assessing unbound brain to plasma ratio as well as pharmacokinetics (PK) and pharmacodynamics (PD) relationship by relating free drug concentration to pharmacological effect in target tissues. In this work, we present a novel microemulsion based capillary electrophoresis (CE) method that enables coupling microemulsion electrokinetic chromatography (MEEKC) to mass spectrometry (MS) for prediction of biopartitioning of CNS drugs in brain tissue. Compared to LC retention based lipophilicity and calculated lipophilicity, a significantly improved correlation between the LogP values obtained from MEEKC retention factors and fraction unbound (fu) in brain tissue was observed for a training set of structurally diverse CNS drugs as well as for a test set of new chemical entities (NCEs). The current online CE/MS/MEEKC technique can also be a potential approach for lipophilicity screening amenable for highly predictive of other ADME-Tox properties of NCEs using the MEEKC partitioning coefficient as a relevant descriptor.