Functions of neuronal circuit are fundamentally modulated by its quality and quantity of connections. Assessment of synapse, the basic unit for a neuronal connection, is labor-intensive and time-consuming in conventional culture systems, due to the small size and the spatially random distribution. In the present study, we propose a novel 'synapse compartmentalization' culture system, in which synapses are concentrated at controlled locations. We fabricated a negative dot array pattern by coating the entire surface with poly-l-lysine (PLL) and subsequent microcontact printing of 1) substrates which mask positive charge of NI (Fc, BSA and laminin), or 2) a chemorepulsive protein (Semaphorin 3F-Fc). By combination of physical and biological features of these repulsive substrates, functional synapses were robustly concentrated in the PLL-coated dots. This synapse compartmentalization chip can be combined with the various high-throughput assay formats based on the synaptic morphology and function. Therefore, this quantifiable and controllable dot array pattern by microcontact printing will be potential useful for bio-chip platforms for the high-density assays used in synapse-related neurobiological studies. (C) 2016 Elsevier Ltd. All rights reserved.