Non-muscle myosin II (NMII)-induced multicellular contractility is essential for development, maintenance and remodeling of tissue morphologies. Dysregulation of the cytoskeleton can lead to birth defects or enable cancer progression. We demonstrate that the Matrigel patterning assay, widely used to characterize endothelial cells, is a highly sensitive tool to evaluate cell contractility within a soft extracellular matrix (ECM) environment. We propose a computational model to explore how cell-exerted contractile forces can tear up the cell-Matrigel composite material and gradually remodel it into a network structure. We identify measures that are characteristic for cellular contractility and can be obtained from image analysis of the recorded patterning process. The assay was calibrated by inhibition of NMII activity in A431 epithelial carcinoma cells either directly with blebbistatin or indirectly with Y27632 Rho kinase inhibitor. Using Matrigel patterning as a bioassay, we provide the first functional demonstration that overexpression of S100A4, a calcium-binding protein that is frequently overexpressed in metastatic tumors and inhibits NMIIA activity by inducing filament disassembly, effectively reduces cell contractility.