In this study, we have demonstrated a method to organize cells in dissociated cultures using engineered chemical clues on the culture surface and determined their connectivity patterns. Although almost all elements of the synaptic transmission machinery between neurons or between neurons and muscle fibers can be studied separately in single-cell models in dissociated cultures, the difficulty of clarifying the complex interactions between these elements makes random cultures not particularly suitable for specific studies. Factors affecting synaptic transmission are generally studied in organotypic cultures, brain slices, or in vivo where the cellular architecture generally remains intact. However, by utilizing engineered neuronal networks, complex phenomenon such as synaptic transmission can be studied in a simple, functional, cell culture-based system. We have utilized self-assembled monolayers (SAMs) and photolithography to create the surface templates. Embryonic hippocampal cells, plated on the resultant patterns in serum-free medium, followed the surface clues and formed the engineered neuronal networks. Basic electrophysiological methods were applied to characterize the synaptic connectivity in these engineered two-cell networks.