Excitotoxicity, over-stimulation of the extrasynaptic glutamate receptors is a pivotal neuronal cell death process underpinning brain damage in acute neurological conditions such as ischaemic stroke and chronic neurodegenerative diseases such as Alzheimer’s disease. Glutamate, the main excitatory neurotransmitter of central nervous system initiates excitotoxicity by causing excessive influx of ions, mostly Ca2+ into neurons and resulting in the activation of different enzymes, such as calpains and caspases. These aberrantly activated enzymes then modify specific neuronal proteins that cause neuronal cell death. How these cellular proteins in neurons undergo proteolytic processing and eventually cause neuronal death, has not been clearly defined. To address this question we employed a high-content N-terminal positional proteomics technique, Terminal Amine Isotopic Labelling of Substrates (TAILS), to identify neuronal proteins that are post-translationally processed and modified in excitotoxicity. This approach involve stable isotope dimethyl labelling of the N-termini of proteins generated by limited proteolysis in primary cortical neurons after glutamate over-stimulation. In the experiment with glutamate treated neurons, we identified 63 proteins as potential key mediators in excitotoxic neuronal death. Further analysis of our study will unveil potential driver signalling pathways directing neuronal death in excitotoxicity.