The development of the central nervous system (CNS) begins before birth and it continues to change and restructure throughout adult life. CNS development relies on inductive factors and signaling molecules secreted by nearby cells that control differentiation of neuronal cells. To study the effect of such molecules on neurons, the mammalian hipppocampus became a well-established and among most used in vitro models. This study is focused on the effect of guidance molecules on neuronal growth cones, the major motile structures guiding both axon and dendrite navigation during the first stages of hippocampal neuronal development in vitro. At this stage, chemotropic guidance of molecular cues can be observed; for instance, netrin molecules function as chemoattractants for hippocampal neurons. On the other hand, semaphorins play part in repulsion of cells. The combination of repulsive and attractive cues allow neurons to reach their targets. Techinques traditionally used in observing molecular guidance are the growth cone collapse assay and growth cone turning assay. In addition, immunofluorescence assays combined with specific neuronal markers can be performed to discriminate between hippocampal neurons and other cell population such as astrocytes, oligodendrocytes, microglia or other non-neuronal cells that are present in the culture. Moreover, the expression of guidance molecules receptors can be investigated. Beside well known, some newly developed techniques will be described, such as localized delivery of guidance molecules by optically manipulated liposomes. Each technique offers different approach and potential to study if, and how, can a certain molecule act as a chemoattractant or chemorepellant during axon guidance, pathfinding and formation of functional neuronal network.