Neurons are probably the most highly polarized cell type and typically develop a single axon and several dendrites. The establishment of a polarized morphology and the functional specialization of axonal and dendritic compartments are essential steps in the differentiation of neurons. Primary cultures of dissociated hippocampal neurons are a widely used system to study the development of neuronal differentiation. In this article, we will describe gain-of-function and loss-of-function approaches that allow us to analyze the role of GTPases in neuronal differentiation.