Abstract Amyotrophic lateral sclerosis (ALS) is a progressive degenerative disease that affects motor neurons. Mutations in the gene SOD1 (superoxide dismutase 1) and in chromosome 9 seem the most prevalent in those affected by the disease. Despite tremendous efforts aimed at identifying contributing factors for ALS, the mechanisms underlying motor neuron death have not yet been fully elucidated and consequently no effective treatment is currently available for ALS. Several clinical trials have been initiated based on drugs selected from animal studies, however, these ultimately failed. Obviously among the possible reasons for such failures is the lack of a proper drug target responsible for the onset and progression of ALS and associated pharmacological tools. In this regard, numerous recent studies clearly suggest that the EphA4 receptor tyrosine kinase is a potential drug target for ALS and that targeting its ligand–binding domain may provide a possible avenue to novel and effective therapeutics. Based on these premises, we have previously recently obtained the first bona fide EphA4 agonistic agents targeting its ligand binding domain, that are brain penetrant and show protection in cellular and animal models of the disease. Our studies aimed at further optimizing and characterizing this series will provide critical pharmacological tools and mechanistic insights on the role of the EphA4 modulation in the progression of ALS, and the data gathered in this study will be critical in supporting the development of these agents into innovative targeted therapeutics for ALS.