The developing spinal cord is a well-established model system widely used to study the signaling pathways and genetic programs that control neuronal/glial differentiation and neural circuit assembly. This is largely due to the relatively simple organization (compared to other CNS regions) and experimental accessibility of the neural tube, particularly in the chick embryo. In vivo transfection of cells within the developing chick neural tube using in ovo electroporation has emerged as a rapid and powerful experimental technique in that (1) transfected factors can be functionally tested in a spatially and temporally controlled manner and (2) the chick embryo provides a physiologically relevant in vivo environment to conduct biochemical studies such as dual-channel luciferase assay, co-immunoprecipitation (co-IP), and Chromatin Immunoprecipitation (ChIP). In this chapter, we will take an in-depth look at the in ovo electroporation system in embryonic chicken spinal cord. In the following chapter, we will continue by examining the use of in ovo electroporation in the dual-channel luciferase assay as an example of its biochemical application.