Springer Science+Business Media New York 2015. All rights reserved. Conventional cell culture plate based neuron-glia coculture methods have limitations in conducting localized physical and biochemical manipulations of cells that allow detailed investigation into axon-glia interactions and spatial regulation of functions. Here, we describe a multi-compartment neuron-glia coculture microsystem platform where axons can be fluidically and physically isolated from neuronal somata and dendrites so that interactions between the axon and glia can be studied in isolation. The multicompartment configuration having six axon/glia compartments allows multiple experimental conditions to be conducted on a single microsystem.