Organotypic tissue slice culture is established from animal or patient tissues and cultivated in an in vitro ecosystem. This technique has made countless contributions to anticancer drug development due to the vast number of advantages, such as the preservation of the cell repertoire and immune components, identification of invasive ability of tumors, toxicity determination of compounds, quick assessment of therapeutic efficacy, and high predictive performance of drug responses. Importantly, it serves as a reliable tool to stratify therapeutic responders from nonresponders and select the optimal standard-of-care treatment regimens for personalized medicine, which is expected to become a potent platform and even the gold standard for anticancer drug screening of individualization in the near future.