Growth cone guidance during development, as well as axonal extension in neural repair and plasticity, is strongly regulated by both attractive (growth-promoting) and repulsive (growth-inhibiting) guidance molecules. The growth cone collapse assay has been widely and successfully used for the identification and purification of molecules that are repulsive to growth cones or inhibit axonal outgrowth. Here we provide a detailed description of the assay, which uses the morphology of the growth cone after exposure to a test protein as the readout. With the modifications detailed in this protocol, this assay can be used for the biochemical enrichment of proteins with a collapsing activity and for the identification of a collapsing activity of a known protein or gene. This assay does not require very specialized equipment and can be established by every lab with experience in neuronal cell culture. It can be completed in 3 d.