It is imperative to analyse brain injuries directly in real time, so as to find effective therapeutic compounds to protect brain injuries by stress. We established a system which could elucidate the real time Ca2⁢ dynamics in an organotypic cultured hippocampal slice by the insults of artificial stress hormone, dexamethasone. The real time Ca2⁢ dynamics could continuously be detected in cornus ammonis 3 (CA3) of the organotypic hippocampus for 8 hours under confocal microscopy. When dexamethasone concentration was increased, the Ca2⁢ was also increased in a dose dependent manner at 1∼100 μM concentrations. Moreover, when the organotypic cultured hippocampal slice was treated with a glutamate receptor antagonist together with dexamethasone, the real time Ca2⁢ dynamics were decreased. Furthermore, we confirmed by PI uptake study that glutamate receptor antagonist reduced the hippocampal tissue damage caused by dexamethasone treatment. Therefore, our new calcium ion dynamics system in organotypic cultured hippocampal slice after dexamethasone treatment could provide real time analysis method for investigation of brain injuries by stress.