Background: Autism Spectrum Disorder (ASD) is a complex neurodevelopmental disorder with a strong genetic and environmental basis. It frequently causes social and communication deficits, as well as repetitive behaviours. Valproic acid (VPA) has been shown to induce autisticlike features in animal models when administered during critical development periods. However, not much is known about its effect on cells to replicate ASD characteristics in vitro. Objective: This review explores in vitro VPA models to elucidate the molecular and morphological characteristics of ASD, emphasizing their potential and proposing directions for future research. Methods: PubMed, SciELO, Embase, Web of Science, and Scopus databases were searched, and 11 studies were included after screening. Results: The studies explored VPA's effects on various cell cultures, including human neural cell lines, primary adult neurons, and primary embryonic neurons. VPA was found to be neurotoxic in a dose- and time-dependent manner, with greater toxicity in immature and undifferentiated cells. In vitro, VPA can influence gene expression, increase oxidative stress, disrupt neurogenesis and synaptogenesis, affect the GABAergic system, and alter critical signaling pathways for brain development and cell differentiation, such as Wnt/β-catenin. Conclusion: In vitro models provide valuable insights into the morpho-molecular alterations induced by VPA and their connection to ASD. These findings highlight the need for further research into VPA's cellular effects to deepen our understanding of its role in ASD pathology.