Advances in microscopy, instrumentation, and image analysis software, combined with improved protocols for acquiring and maintaining primary neuronal cultures, have made high content analysis (HCA) with primary neurons a routine procedure in many labs. Additional improvements in robotics and automation allow the high-throughput application of HCA in high-content screening (HCS). Given its multiplexed nature, HCS provides an unprecedented amount of multiparametric kinetic and morphogenic information from cells. It holds promise for better understanding of molecular signaling networks, and for easing the bottleneck in the drug discovery process, particularly relating to neurological disorders. Here we describe protocols for preparing different neuronal types and discuss advantages, challenges, pitfalls, and detailed workflows for utilizing them in HCS with various perturbagens.