The emergence of patient-derived tumor organoids (PDOs) has revolutionized preclinical research and precision medicine in oncology. PDOs, derived from patient tumor samples, faithfully recapitulate the histological and molecular characteristics of the original tumor, making them invaluable tools for research and clinical practice. This review provides an overview of methods for establishing PDOs and their applications in cancer research, from basic research to the identification of new targets and preclinical validation of anticancer compounds. PDOs are particularly useful for studying tumor heterogeneity, mechanisms of resistance to treatments, and the development of new therapeutic strategies. They can be used to screen therapeutic molecules, identify new therapeutic targets, and define predictive molecular signatures for treatment response. However, challenges such as representativeness, establishment success rate, assay speed, and the lack of a complete tumor microenvironment must be addressed to optimize their clinical implementation. Technological advancements, including coculture systems and high-throughput screening, are ongoing to overcome these limitations and fully realize the potential of PDOs in precision oncology.The emergence of patient-derived tumor organoids (PDOs) has revolutionized preclinical research and precision medicine in oncology. PDOs, derived from patient tumor samples, faithfully recapitulate the histological and molecular characteristics of the original tumor, making them invaluable tools for research and clinical practice. This review provides an overview of methods for establishing PDOs and their applications in cancer research, from basic research to the identification of new targets and preclinical validation of anticancer compounds. PDOs are particularly useful for studying tumor heterogeneity, mechanisms of resistance to treatments, and the development of new therapeutic strategies. They can be used to screen therapeutic molecules, identify new therapeutic targets, and define predictive molecular signatures for treatment response. However, challenges such as representativeness, establishment success rate, assay speed, and the lack of a complete tumor microenvironment must be addressed to optimize their clinical implementation. Technological advancements, including coculture systems and high-throughput screening, are ongoing to overcome these limitations and fully realize the potential of PDOs in precision oncology.