The increasing prevalence of neurodegenerative diseases such as Parkinson’s and Alzheimer’s necessitate more advanced neural circuit models for research and drug development. Traditional rodent models fail to replicate human brain complexity, leading to high late-stage clinical trial failure rates. This study presents the development and optimisation of a humanised microfluidic in vitro neuronal model using compartmentalised microfabricated devices to culture SH-SY5Y neurons. The differentiation protocol was refined to promote long, functional neurons suitable for forming unidirectional circuits. Electrophysiological assessment through calcium imaging and multi-electrode array (MEA) recordings confirmed neuronal activity. The successful integration of SH-SY5Y neurons into PDMS devices demonstrates the potential of this model for long-term studies of neural circuit behaviour. Further developments will enhance neuronal connectivity and disease modelling capabilities.