New in vitro technologies that assess neuronal excitability and the derived synaptic activity within a controlled microenvironment would be beneficial for the characterisation of compounds proposed to affect central nervous system (CNS) function. Here, a microfluidic system with computer controlled compound perfusion is presented that offers a novel methodology for the pharmacological profiling of CNS acting compounds based on calcium imaging readouts. Using this system, multiple applications of the excitatory amino acid glutamate (10 nM-1 mM) elicited reproducible and reversible transient increases in intracellular calcium, allowing the generation of a concentration response curve. In addition, the system allows pharmacological investigations to be performed as evidenced by application of glutamatergic receptor antagonists, reversibly inhibiting glutamate-induced increases in intracellular calcium. Importantly, repeated glutamate applications elicited significant increases in the synaptically driven activation of the adjacent, environmentally isolated neuronal network. Therefore, the proposed new methodology will enable neuropharmacological analysis of CNS active compounds whilst simultaneously determining their effect on synaptic connectivity.