Despite the development of various transfection methods, the transfection of post-mitotic cells, including neurons, poses a challenging task. Nucleofection, a specialized form of electroporation described in this unit, achieves high transfection efficiencies in primary mammalian neurons, such as hippocampal neurons, while simultaneously maintaining high cell viability. Therefore, it allows for biochemical analyses that rely on large numbers of transfected cells. The recently developed 96-well shuttle system described in this unit further permits the transfection of up to 96 different constructs in a single experiment. This opens up the possibility for large-scale experiments in primary neurons, such as shRNA-mediated knock-down of a wide range of target genes.