Advancement of therapeutics for neurodegenerative diseases like amyotrophic lateral sclerosis (ALS) has been predominantly hampered by the dearth of relevant disease models. Despite numerous animal models, significant challenges remain in correlating these with human disease complexities. In this study, the ALS model was created using amniotic membrane-derived mesenchymal stem cells (AM-MSCs) which were differentiated into motor neurons (MN) with specific MN induction media and transiently transfected with mutated human SOD1 G93A plasmid to induce ALS-like condition. Characterization included gene expression analysis, immunocytochemistry, flow cytometry, and Western blot. Functional assays assessed the extent of degeneration and model efficiency. AM-MSCs demonstrated multipotency and were positive for MSC markers. Upon differentiation, the expression of MN markers like MNX1, Olig2, and ChAT were found to be elevated. SOD1 G93A overexpression, downregulated MN markers, upregulated NURR1 gene, reduced acetylcholine (ACh), reduced glutathione, and elevated oxidative stress markers. This robust in-vitro ALS model derived from AM-MSCs offers an alternative to animal models to provide an efficient and cost-effective platform to conduct rapid drug screening.