A series of new 8-amino-1,4-benzoxazine derivatives 5a-o was synthesized and examined for their intrinsic cytotoxicity and their capacity to inhibit oxidative stress-mediated neuronal degeneration in neuronal cell cultures. In particular, substituents effects at the 3- and 8-positions of the 1,4-benzoxazine ring were investigated by in vitro evaluation. In this aim, 3-alkyl substituents seemed to be essential for efficient neuroprotective activity. Furthermore, within the sub-series of substituted 3-alkyl benzoxazines, the most active derivatives were those bearing a 8-benzylamino substituent. From the combined results of both toxicity and neuroprotection expressed in terms of the safety index, 8-benzylamino-substituted-3-alkyl-1,4-benzoxazines were identified as the most promising compounds owing to their potent neuroprotective activity, without the manifestation of intrinsic cytotoxicity.