In vivo electroporation is a method for delivery of plasmids and other oligonucleotide reagents that offers precise temporal control. In zebrafish, in vivo electroporation is particularly well-suited to delivering green fluorescent protein (GFP) expression vectors to the developing central nervous system. This protocol describes a modification of in vivo electroporation that can be used to specifically target the developing optic tectum of zebrafish embryos beginning at 24 h post-fertilization (hpf). The electroporation electrodes required for this approach can be constructed easily from relatively inexpensive materials. Microinjection of plasmid DNA to the midbrain ventricle followed by precise positioning of the electroporation electrodes allows for the targeting of developing neurons in only one hemisphere of the optic tectum. Using this protocol, the optic tectum can be effectively targeted in a high percentage (79%) of expressing embryos. This method can also be used to simultaneously deliver expression vectors and loss-of-function reagents, which can provide precise temporal control of the knockdown of gene function.