Tissue culture has emerged as a cornerstone in modern toxicology, offering controlled in vitro systems to study cellular responses to chemical, environmental, and pharmaceutical agents. By cultivating primary cells or established cell lines under defined conditions, researchers can investigate cytotoxicity, metabolic activity, and molecular stress responses without the ethical and logistical limitations of animal models. Recent advances, including 3D organoid cultures, co-culture systems, and microfluidic “organ-on-a-chip” platforms, provide more physiologically relevant models that closely mimic tissue architecture and intercellular interactions. These innovations allow for more accurate prediction of human toxicological outcomes, high-throughput screening of compounds, and mechanistic studies of cellular pathways affected by toxins. Integrating tissue culture techniques with genomic, proteomic, and imaging analyses is transforming toxicology research, facilitating safer drug development and better understanding of environmental health risks.