The structures of the tissue microenvironment influence cell function and behavior, both in physiological and pathological conditions. Cells experience and integrate a multitude of mechanical and physical cues from this 3D tissue microenvironment to adapt to organismal development. In turn, they respond by exerting forces, regulating their shape, internal cytoskeletal tension, and elastic modulus. Disruption of the cellular forces, as well as variations in subcellular mechanical properties, can lead to altered pathophysiological conditions and the onset of diseases i.e., cancer. Diseases like cancer are characterized by dramatic changes in cell and tissue mechanics, and dysregulation of forces at the cell and tissue level can activate mechanosensing to compromise tissue integrity and function and promote disease progression. Cells can move by exerting traction forces to their environment. These can be assessed by using microfabricated substrates and improved computational approaches, enabling us the characterization of biomechanical forces generated by single cells cultured in defined microenvironments. This provides valuable insights into the malignancy of cancer cells. However, the conventional microfabricated structures i.e., 2D substrates are far from matching the complexities present in vivo. Hence, there is a need for biomimicking the natural surfaces for cellular applications. We here show the traction forces induced by single glioblastoma cells in three-dimensional (3D) tumor microenvironment-inspired scaffolds i.e., collagen. The dimensionality of cell culture influences cell motility and cellular interaction with the surrounding cells and ECM. As cells grown on 2D scaffolds, adapt to the artificial environment and may no longer display characteristics of the original tumor. An attempt to develop 3D tumor microenvironment-inspired scaffolds and their functionality in unraveling the role of microenvironment on tumor cell behaviors are also examined. Characterizing the cues involved in glioblastoma cell migration could enable the scientific and medical community to develop better strategies to understand and treat brain cancer.