In ovo RNA interference (RNAi) is a method for silencing a gene of interest using a combination of in ovo injection and electroporation in avian embryos. Here we describe gene silencing in the developing spinal cord, but the procedure can easily be adapted to other parts of the nervous system. Double-stranded RNA (dsRNA) derived from the gene of interest is injected into the developing spinal cord of the chicken embryo, and is followed by electroporation to allow for the uptake of the dsRNA. With this method, temporal as well as spatial control of gene silencing is possible. The time point of injection should be chosen according to the expression profile of the gene or the half-life of the protein. Proteins with slow turnover may require RNAi at earlier stages, ideally before the onset of gene expression. The electroporation parameters can be adjusted such that only a specific population of neurons is targeted in the spinal cord.