Cerebral ischemia or stroke, an acute neurological injury lacking an effective therapy, is the second leading cause of death globally. The unmet need in stroke research is to identify viable targets and to understand their interplay during the temporal evolution of ischemia/reperfusion (I/R) injury. Here we report a temporal signature of the ischemic hemisphere revealed by the isobaric tag for relative and absolute quantification (iTRAQ)-based 2D-LC-MS/MS strategy in an in vivo middle cerebral artery occlusion (MCAO) model of focal cerebral I/R injury. To recapitulate clinical stroke, two hours of MCAO was followed by 0, 4, and 24 h of reperfusion to capture ischemia with an acute and subacute durations of reperfusion injury. The subsequent iTRAQ experiment identified 2242 proteins from the ischemic hemisphere with <1.0% false discovery rate. Data mining revealed that (1) about 2.7% of detected proteins were temporally perturbed having an involvement in the energy metabolism (Pygb, Atp5b), glutamate excitotoxicity (Slc1a3, Glud1), neuro-inflammation (Tf, C3, Alb), and cerebral plasticity (Gfap, Vim, Gap43); (2) astrocytes participated actively in the neurometabolic coupling underlining the importance of a cerebro-protective rather than a neuro-protective approach; and (3) hyper-acute yet progressive opening of the blood brain barrier (BBB), accompanied by stimulation of an innate immune response and late activation of a regenerative response, which provides an extended therapeutic window for intervention. Several regulated proteins (Caskin1, Shank3, Kpnb1, Uchl1, Mtap6, Epb4.1l1, Apba1, and Ube1x) novel in the context of stroke were also discovered. In conclusion, our result supports a dynamic multitarget therapy rather than the traditional approach of a unilateral and sustained modulation of a single target to address the phasic regulation of an ischemic proteome.