Stroke is a clinically defined neurological syndrome characterized by rapidly progressing symptoms and signs of focal loss of cerebral function. The initiation, propagation, and maturation of ischemic stroke are associated with de novo expression of multiple genes in endogenous brain tissues and infiltrated inflammatory cells. This chapter provides an overview for the use of state-of-the-art molecular biological approaches to investigate de novo gene expression in animal models of focal stroke, including subtractive cDNA library screening, mRNA differential display, suppression subtractive hybridization, representational difference analysis, serial analysis of gene expression, and microarrays. Identification of stroke-related gene expression will facilitate the understanding of the molecular basis of stroke pathogenesis and may provide a novel therapeutic intervention of the disease.