Summary Genetic manipulations of dissociated rodent neurons provide translatable in vitro models for disease-driven phenotypes. Cilia are cellular antenna with a role in neuronal maturation and function often perturbed in neurodevelopmental disorders. Efforts for automated imaging of these microscopic protrusions are crucial given the role of cilia in the brain. We developed a cell-based assay to monitor cilia in rat hippocampal neurons using lentiviral-mediated shRNA-based gene silencing. This optimized platform can be used for high-throughput cilia imaging, disease modeling, and drug screening. For complete details on the use and execution of this protocol, please refer to Di Nardo et al. (2020) .