Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the progressive loss of upper and lower motor neurons, leading to muscle weakness, paralysis, and respiratory failure. ALS exhibits considerable clinical heterogeneity, with varying onset sites, progression rates, and severity of symptoms. The pathological hallmarks of ALS include motor neuron degeneration, TAR DNA-binding protein 43 (TDP-43) proteinopathy, and neuroinflammation, with additional contributions from oxidative stress, mitochondrial dysfunction, and excitotoxicity. While genetic factors play a key role in familial ALS cases, sporadic ALS (sALS) accounts for most diagnoses. Environmental influences such as toxin exposure and traumatic brain injury may also be implicated. Despite significant research advances, ALS remains incurable, and effective treatments are lacking due to its complexity and variability. Experimental models are crucial for deciphering ALS pathophysiology and developing targeted therapies. Two-dimensional (2D) cell cultures, including primary neuron cultures and neuroblastoma cell lines, have been widely employed to study ALS-related genetic mutations, protein aggregation, and cellular dysfunction. While these models provide valuable insights, they lack the complexity of cellular interactions observed in vivo. Organotypic slice cultures helped overcome some of the limitations of 2D cultures. Three-dimensional (3D) models, such as tissue-engineered constructs, organoids, and neuromuscular co-cultures, offer improved physiological relevance by mimicking the cellular microenvironment and interactions between neurons, glial cells, and muscle tissue. Induced pluripotent stem cell (iPSC)-derived models enhance ALS research by enabling patient-specific investigations and capturing genetic heterogeneity and disease variability. Understanding ALS’s diverse manifestations through robust experimental models is essential for biomarker discovery and therapeutic development, ultimately improving patient prognosis and care. Despite their advantages, in vitro models face challenges related to reproducibility, cost, and limited recapitulation of ALS complexity. However, continuous advancements in bioengineering and stem cell technology hold promise for refining these models. Moreover, experimental models for ALS also face significant translational challenges, particularly for sALS. Existing models, predominantly based on familial ALS mutations, fail to capture the complexity and heterogeneity of sALS, limiting their predictive value for the full disease spectrum. Advances in gene-editing technologies like CRISPR/Cas9, patient-derived iPSC models, and integrative approaches combining different cell modeling methods offer promising alternatives. This review categorizes and evaluates available ALS models, highlighting their strengths and limitations. It also aims to highlight the limitations of current models and explore emerging strategies to improve disease modeling, enhance translational potential, and bridge the gap between preclinical research and clinical applications.