AbstractThe mechanical characterization of cell spheroids, one of the most widely used 3D biology models in vitro, is a hotspot of current research on the role played by the mechanical response of cells and tissues. The techniques proposed so far in the literature, while providing important scientific insights, require specialized equipment and technical skills that are not usually available in cell biology facilities. Here, an innovative rheo‐optical compression assay is presented: it is based on microscopy glass coverslips as the load is applied to cell spheroids in standard cell culture plates and on image acquisition with an optical microscope or even a smartphone equipped with adequate magnification lenses. Mechanical properties can be simply obtained by correlating the applied load to the deformation of cell spheroids measured by image analysis. The low‐cost, user‐friendly features of the proposed technique can boost mechanobiology research making it easily affordable to any biomedical lab equipped with cell culture facilities.