Cancer is one of the most severe diseases diagnosed in millions of people worldwide each year. Despite many studies, there is insufficient information on how tumor formation prevents cancer treatment development. Although clinical trials are the most effective way to examine tumor formation and test anti-tumor drugs, ethical and safety limitations prevent this method from being widely used. This study aims to test the cellular behavior of different cancer cell lines on the platform obtained from decellularized adipose tissue in vitro. Detergent-based decellularization protocol applied to adipose tissue and cancer cell lines were seeded on obtained extracellular matrixes. Cell viability and apoptosis were observed by MTT assay and Acridine orange/Propidium iodide staining, respectively. Also, cell-cell and cell-matrix interactions were investigated via pan-cadherin immunostaining. All cancer cell lines were also seeded in a cell culture dish to compare three-dimensional culture results with two-dimensional culture. As a result, the decellularization protocol allowed the original structure of the tissue scaffold to be preserved. According to cell viability analysis and immunocytochemical staining results, T98G and Hep3B cell lines were observed to have higher adhesion and viability potential than the WiDr cell line on the tissue matrix obtained. With this study, it can be said that different cancer cells have different behaviors on decellularized matrixes. Finally, developing in vitro models as a more economical, scalable, and reproducible way to test drugs and therapeutics is crucial for successful clinical translation.