Neurotoxicity, the detrimental impact of substances on the nervous system, necessitates a comprehensive exploration of its underlying cellular and molecular mechanisms. This chapter highlights the multifaceted applications of flow cytometry in advancing our understanding of neurotoxicity. Flow cytometry has become a pivotal tool in the realm of neurotoxicity research, enabling precise assessments across various dimensions. It serves as a linchpin for evaluating cell viability and apoptosis, offering insights into the effects of neurotoxic agents on neural cell populations. Moreover, flow cytometry facilitates the isolation and purification of specific neuronal subtypes, aiding in the meticulous analysis of cells afflicted by neurotoxic insults. The technique’s utility extends to probing intracellular signalling pathways, allowing the examination of protein phosphorylation changes critical to neurotoxicity mechanisms. Additionally, it empowers the quantification of oxidative stress levels in neural cells, a hallmark of neurotoxicity. Neuroinflammation, a substantial contributor to neurotoxicity, is scrutinized through flow cytometry’s ability to characterize immune responses in the central nervous system. The analysis of immune cell populations and cytokine secretion provides invaluable insights into the inflammatory processes accompanying neurotoxic insults. Flow cytometry further illuminates neural stem cell behaviour, facilitating the assessment of differentiation, proliferation, and survival in the presence of neurotoxic agents. It assists in quantifying alterations in neuronal protein expression, potentially identifying biomarkers or therapeutic targets. Additionally, it enables the evaluation of mitochondrial function, a critical facet of neurotoxicity, through the measurement of mitochondrial membrane potential and function. Lastly, flow cytometry plays a pivotal role in high-throughput drug screening, aiding in the identification of compounds with neuroprotective or neurotoxic properties. In conclusion, flow cytometer’s diverse applications have established it as an indispensable tool for delving into the intricate facets of neurotoxicity, offering an encompassing perspective on cellular and molecular responses to neurotoxic agents. Its versatility in assessing cell viability, signalling pathways, immune responses, and stem cell behaviour positions it as an indispensable asset in neurotoxicity research and therapeutic development endeavours.