Human cancer cell lines are invaluable tools in cancer research, playing a pivotal role in signalling the complexities of tumor biology, investigating signalling pathways, drug discovery, and exploring immunotherapy approaches. Despite their limitations, they have significantly contributed to our understanding of cancer and have been instrumental in the development of new treatments. The emergence of advanced technologies, such as organoid cultures, organ-on-a-chip systems, and single-cell analysis, holds great promise for overcoming the limitations of traditional cell line models and providing more physiologically relevant platforms. These advancements have the potential to revolutionize cancer research, enhance our understanding of tumor heterogeneity, and pave the way for personalized medicine. With ongoing improvements in standardization and quality control, human cancer cell lines will continue to be indispensable in driving future advancements and improving patient outcomes in the battle against cancer.