Cortical neurons grown on multielectrode array (MEA) chips have been shown to be a valuable alternative method to study electrophysiological properties of the central nervous system neurons and to perform functional toxicological screening. Here we studied the effects of binary mixtures on neuronal networks cultured on MEAs. We have considered compounds with similar and different mode-of-action (MoA) to characterize and assess their combined effects. Individual and binary mixture dose-response curves based on spontaneous neuronal activity have been generated and the IC50 has been considered as the end-point for neurotoxicity assessment. The two classical approaches of mixtures toxicity studies: concentration addition (CA) and independent action (IA) have been applied to compare calculated and experimental results. Nuclear magnetic resonance (NMR) spectroscopy has been employed to confirm no chemical reaction or complexation between mixtures components. The results suggest that both CA and IA are able to predict the toxicity of the mixture and that the combination of in vitro test methods with theoretical dose-response models has a strong potential as an alternative tool for the prediction of mixtures neurotoxicity. © 2012 Elsevier Ireland Ltd.