A popular approach to study living neurons involves the preparation of dissociated cultures. If isolated from neonatal or embryonic animals, neurons survive their removal and subsequent dissociation procedures. They grow processes on appropriate substrates, acquire mature neuronal morphology and polarity, and form functional synapses that resemble those of neurons developing in vivo. On the other hand, dissociated neurons produce neural circuits which differ from native circuits. While many protocols have been developed to grow multiple types of neurons, this chapter focuses on one major protocol: the preparation of neonatal rat hippocampal neurons. It involves several steps including the meticulous preparation of coverslips and stock solutions, dissection of the hippocampi, cell seeding, and the long-term maintenance of the cultures. While dissociated neuronal cultures are used by many and can be routinely maintained for several weeks, they deserve particular attention through daily observations and rigorous laboratory practice.