Metabolomics research plays a critical role in understanding the molecular foundations of diseases, including cancer. This study provides a comprehensive protocol for extracting metabolites from the colorectal cancer cell line SW620, comparing the processes in both 2D and 3D cell cultures. The protocol outlines key procedures for culturing cells, handling multicellular tumor spheroids, and extracting metabolites in both culture models. These models provide distinct insights into tumor biology, highlighting differences between the two culture systems. With 3D cell cultures offering a more accurate simulation of the in vivo tumor environment due to their three-dimensional architecture. This architecture influences key cellular processes, such as gene expression, nutrient availability, and protein synthesis, leading to variations in metabolite concentrations between 2D and 3D models and reflecting the biological complexity of tumors in vivo. The findings underscore the limitations of traditional 2D cultures in replicating tumor behavior and support the growing use of 3D models in cancer research. This protocol serves as a valuable resource for researchers seeking to obtain high-quality metabolomic data from colorectal cancer cell lines. By employing both 2D and 3D culture methods, scientists can gain a deeper understanding of how tumor cells metabolically adapt to their environment, as well as more accurate insights into cancer progression and treatment responses. In conclusion, this protocol provides a foundation for future studies on colorectal cancer and may aid in the development of more effective therapeutic strategies.