Excitotoxicity, initiated by over-stimulation of ionotropic glutamate receptors (iGluRs), is a major pathological process directing regulated necrosis of neurons in both acute and chronic neurological disorders. Upon over-stimulation, iGluRs allow massive influx of calcium ions into the affected neurons, leading to over-activation of two groups of neurotoxic calcium-dependent enzymes: (i) the cysteine proteases calpains, which catalyse limited proteolysis of specific neuronal proteins to modulate their functions and (ii) neuronal nitric oxide synthase (nNOS), which generates excessive NO to induce oxidative damages. The calpain-proteolysed proteins and the NO-induced oxidative damages in turn modulate the activities of proteases, protein kinases and phosphatases to perturb the expression and phosphorylation of specific neuronal proteins. Presumably, these perturbed proteins form signalling networks that direct neuronal necrosis. To define these signalling networks, we aim to identify the calpain substrates and the perturbed proteins in neurons undergoing excitotoxic cell death. Using the Terminal Amine Isotopic Labelling of Substrates (TAILS) proteomics method, we identified the exact sites of cleavage in ~300 neuronal proteins proteolytically processed by calpains and other proteases activated in neurons undergoing necrotic death. Additionally, using the stable isotope dimethyl labelling method, we definitively identified ~1300 neuronal proteins and ~1000 phosphosites in neurons undergoing excitotoxic cell death. Among them, around 150 neuronal proteins exhibited dynamic changes in abundance and/or phosphorylation levels in response to glutamate over-stimulation. Bioinformatic analysis revealed that some of the calpain substrates and neuronal proteins exhibiting significant changes in phosphorylation levels form distinct signalling networks. Using biochemical approaches, we found that some components of the predicted signalling networks induce neuronal death by aberrant regulation of key protein kinases critical to neuronal survival. Taken together, our findings illustrate how results of quantitative proteomic analyses can form the conceptual framework for investigation to define the molecular mechanism governing regulated necrosis of neurons.