Classically, primary sensory neuron cultures obtained from the DRG have been used as a model to evaluate neurite growth in vitro. Primary sensory neurons are easily cultured, either dissociated or from explants, from embryonic to adult ages. In contrast, culture of motoneurons is much more complex and limited to the embryonic ones or to postnatal organotypic cultures by using membrane culture inserts. Here we describe a protocol of an easy in vitro assay to culture postnatal rodent spinal cord organotypic slices and DRG explants in 3D collagen matrices that are permissive for neuritogenesis. The main aim of this in vitro assay is to have a similar setting for both types of neurons that allows the measurement and comparison of positive or adverse events on neurite growth of motor and sensory neurons. The matrix can also be modified by adding trophic or tropic factors, cells, or other agents. Immunohistochemistry of the explants and the slices is needed to specifically label myelinated fibers and fairly compare the growth of myelinated primary sensory neurons and motoneurons, as well as neuronal survival.