Background: Cerebrovascular accident is the most common and devastating disorders in old age group. The diagnosis of stroke remains a clinical one, with confirmatory evidence obtained through neuroimaging. Neurobiochemical markers have gained special importance in the determination of brain damage resulting from acute stroke. Aim: In this study, we aimed to evaluate serum S-100 protein in blood samples from patients with acute ischemic stroke and investigate the relationship of serum S-100 protein level with the involved territory and size of the lesion. Methods: This was a prospective observational study conducted among 94 patients of acute ischemic stroke admitted to the Medicine Department within 48 h. Serum sample was collected within 48 h and was sent for measurement of serum S-100 protein level. Patients were classified according to involved territory as anterior cerebral artery (ACA), middle cerebral artery (MCA), posterior cerebral artery (PCA), and more than one territory and correlate it with S-100 protein level. All patients had cranial computerized tomography scan and magnetic resonance imaging in the first 48 h. Neurological examination was done with National Institute of Health Stroke Scale in acute stage and Rankin scale at the time of discharge. Results: Serum S-100 protein levels were significantly higher and maximum in multiple territory involvements followed by MCA, PCA, and ACA infarct. Conclusions: As serum S-100 protein level correlates with the involved territory or infarct size, we can predict the involved territory with the level of S-100 protein.