The mechanical properties of articular cartilage serve as important measures of tissue function or degeneration, and are known to change significantly with osteoarthritis. Interest in small animal and mouse models of osteoarthritis has increased as studies reveal the importance of genetic background in determining predisposition to osteoarthritis. While indentation testing provides a method of determining cartilage mechanical properties in situ, it has been of limited value in studying mouse joints due to the relatively small size of the joint and thickness of the cartilage layer. In this study, we developed a micro-indentation testing system to determine the compressive and biphasic mechanical properties of cartilage in the small joints of the mouse. A nonlinear optimization program employing a genetic algorithm for parameter estimation, combined with a biphasic finite element model of the micro-indentation test, was developed to obtain the biphasic, compressive material properties of articular cartilage. The creep response and material properties of lateral tibial plateau cartilage were obtained for wild-type mouse knee joints, by the micro-indentation testing and optimization algorithm. The newly developed genetic algorithm was found to be efficient and accurate when used with the finite element simulations for nonlinear optimization to the experimental creep data. The biphasic mechanical properties of mouse cartilage in compression (average values: Young’s modulus, ; Poisson’s ratio, 0.20; and hydraulic permeability, ) were found to be of similar orders of magnitude as previous findings for other animal cartilages, including human, bovine, rat, and rabbit and demonstrate the utility of the new test methods. This study provides the first available data for biphasic compressive properties in mouse cartilage and suggests a promising method for detecting altered cartilage mechanics in small animal models of osteoarthritis.