Collagen hydrogels have been widely used to model biological systems and examine cell behavior in vitro. Of increasing interest is how cells affect the mechanical characteristics of their surrounding matrix and vice versa over long culture periods. In this study, the change in mechanical properties of collagen hydrogels embedded with human corneal fibroblasts was examined over a 6-week culture period using a novel online spherical indentation system. The elastic modulus of the hydrogels was found to increase during the first 2 weeks of culture and decrease after 4 weeks in culture. The effects of actin polymerization and matrix metalloproteinase inhibitors were also examined, which verified some mechanisms involved in the alteration of the mechanical properties such as cell tensile forces and other potential factors. This online monitoring technique demonstrates the ability to examine the mechanical properties of cell-seeded constructs in response to the culture environments—in particular, the response to the addition of drugs or chemical reagents—which will provide a useful tool in studying the mechano-feedback loop between cells and their surrounding matrix.