Fos protein, an immediate early gene product, is widely used as a biological marker of neural excitation in the neuropharmacology research. Specifically, mapping analysis of Fos expression is a useful method to identify brain regions related to disease conditions (e.g., epilepsy, emotional disorders and cognitive impairments) and responses to various pathophysiological stimuli (e.g., pain, body temperature, stress, and drug treatments). Immunohistochemical staining of Fos protein can be performed by the conventional avidin-biotinylated-horseradish peroxidase complex (ABC)-diaminobenzidine (DAB) method and the counting of Fos-immunoreactivity positive neurons in the regions of interest allows topographical and quantitative analysis of neuronal network activation. In this chapter, we introduce general methods to analyze Fos protein expression, providing essential information for its application to in vivo neuropharmacology research on central nervous system (CNS) disorders and drug actions. Accurate and reliable analysis of Fos expression can help our understanding of the mechanisms underlying the pathogenesis and treatment of CNS disorders.