Low-density cultures of embryonic rat hippocampal neurons have been widely used to investigate localization and function of neuronal proteins using immunocytochemistry and electrophysiology. These cultures provide a relatively homogeneous population of hippocampal pyramidal neurons and interneurons compared to post-natal mixed neuron/glial cultures from hippocampus, cerebral cortex, and cerebellum. However, the limited quantity of neurons and the difficulty in harvesting adequate amounts makes biochemical analyses of endogenous neuronal proteins in these low-density cultured neurons difficult. Here, we provide detailed methods to prepare cultures of embryonic rat hippocampal neurons suitable for biochemical analyses of both endogenously and exogenously expressed proteins. The procedures described here are also suitable for comprehensive studies of expression, localization, post-translational modification, and function of neuronal proteins in the same neuronal culture system.