Microdialysis has been the only direct method of continuously measuring the unbound drug concentrations in extracellular fluid at a specific brain region with respect to time in the same animal. However, not every compound is suitable for microdialysis system as demonstrated by their inconsistent “by gain” and “by loss” in-vitro microdialysis probe recoveries leading to over- or under- estimated in-vivo concentrations. Therefore, our current study was proposed aiming to develop simple exclusion criteria for drug candidates that are not suitable for microdialysis system investigation. Through literature research, the properties ((LogP, pKa, water solubility and unbound fraction in plasma and brain) of drugs that have been reported for microdialysis studies were summarized. The exclusion criteria were developed by evaluating the impact of such properties on the consistency of in-vitro “by gain” and “by loss” recoveries of microdialysis probe. As a result, forty-five compounds were identified from literatures, among which doxorubicin, docetaxel, omeprazole, donepezil and phenytoin were found to have inconsistent in-vitro “by gain” and “by loss” microdialysis probe recoveries and subsequently selected for the exclusion criteria analysis. It was found that compounds with limited water solubility (less than 1 g/L) and unbound fraction in plasma (f_u,plasma less than 30%) and brain homogenate (f_u,brain less than 10%) were more likely to have inconsistent “by gain” and “by loss” microdialysis probe recoveries. Our proposed exclusion criteria were further validated using carbamazepine (limited water solubility only), DB213 (limited f_u,brain only) and piperine (both limited water solubility and limited f_u,plasma, f_u,brain). Our current proposed exclusion criteria will help excluding the CNS drug candidates that are highly unlikely suitable for brain microdialysis approach leading to a better success rate in brain microdialysis approach development.