This study investigates the feasibility of a different new approach to determining the microleakage volume associated with dental restorations (Class V cavity restorated with glass ionomer cement + high copper amalgam) and the relative marginal adaptation deficiency of dog, bovine and human permanent teeth in in vitro conditions. Also researched is the appropriateness of using dog and bovine teeth in in vitro studies rather than human teeth. Our method utilizes the molecular adsorption characteristics of methylene blue. Within the framework of this study, 60 permanent teeth (20 human, 20 dogs and 20 bovine) were used. These groups were evaluated statistically, of which indicated no statistically significant differences (p > 0.05). It was also concluded that this preliminary investigation showed that the new microleakage volume measurement method may be a valuable new technique for the in vitro study of microleakage dynamics around dental restorations.