Objective : The water extract of Danguiyonghoihwan (DGYHW) has been traditionally used in treatment of tinnitus in Oriental Medicine. However, little is known about the mechanism by which DGYHW rescues auditory neuronal cells from injury damages. Therefore, in this study I effort to elucidate the mechanism of the cytoprotective effect of the DGYHW extract on glutamate-induced auditory sensorineuronal cell death. Methods : I determined the elevated cell viability by DGYHW extract on glutamate-induced auditory sensorineuronal cell death. Glutamate induced neuronal damage in oranotypic explant culture also, glutamate decreased cell viability on VOT-33 cells but pretreatment with DGYHW inhibited cell death. Results : One of the main mediator of glutamate-induced cytotoxicity was known to generation of reactive oxygen species (ROS). Pretreatment with DGYHW inhibited this ROS generation from glutamated-stimulated VOT-33 cells. Also, I identified that the ROS-induced DCF-DA green fluorescence is reduced by DGYHW pretreatment. The critical markers of apoptotic cell death were cleavages of procaspase-3 protease protein. So I checked the expression level and cleavage of procaspase-3 protease protein. Glutamate-treated VOT-33 cells were shown to have cleavage of procaspase-3 protease proteins and following reduction of expression of these proteins. But I found that pre-treatment with DGYHW protects glutamate-induced changes of biochemical marker protein, caspase-3. Conclusion : These findings indicated that DGYHW may prevent cell death from glutamate induced VOT-33 cell death by inhibiting the ROS generation and modulation of protein expressions in procaspase-3, catalase and Bcl-2.