Methods to compartmentalize neurons allow distinct neuronal segments (i.e., cell bodies, axons, dendrites, or synapses) to be accessed, visualized, and/or manipulated. Compartmentalization has resulted in multiple studies that would not otherwise be possible in vivo or in traditional random cultures, such as investigations of axonal transport, biochemical analysis of axons, and axonal injury/regeneration. Chambers for compartmentalizing neurons were first developed for long projection peripheral neurons in the 1970s using machined Teflon dividers and relied on manually applied grease layers to spatially and fluidically separate distal axons from their cell bodies. More recently microfabrication and soft lithography techniques have been used to create compartmentalized microfluidic platforms, relying on microgrooves contained within a solid barrier through which axons and dendrites are able to extend, but not their cell bodies. These platforms are unique in their ability to culture central nervous system (CNS) neurons and allow high-resolution live imaging. These microfluidic platforms have allowed new investigations of axonal and synaptic biology in the CNS. Moreover, these microfluidic platforms offer improvements for other neural cell and tissue preparations. In this review we discuss traditional methods for compartmentalization, compartmentalized microfluidic platforms, and their use for neurobiology. Lastly, we discuss the use of these platforms for defining and manipulating synapses both pharmacologically and by electrical stimulation and recording.