Cultured neuronal networks with a controlled structure have been widely studied as an in vitro model system to investigate the relationship between network structure and function. However, most cell culture techniques lack the ability to control network structures during cell cultivation, making it difficult to assess functional changes induced by specific structural changes. In this study, we present an in situ manipulation platform based on gold-nanorod-mediated thermoplasmonics to interrogate an in vitro network model. We find that it is possible to induce new neurite outgrowths, eliminate interconnecting neurites, and estimate functional relationships in matured neuronal networks. This method is expected to be useful for studying functional dynamics of neural networks under controlled structural changes.