With the onset and progression of osteoarthritis (OA), articular cartilage (AC) mechanical properties are altered. These alterations can serve as an objective measure of tissue degradation. Although the mouse is a common and useful animal model for studying OA, it is extremely challenging to measure the mechanical properties of murine AC due to its small size (thickness < 50 μm). In this study, we developed novel and direct approach to independently quantify two quasi-static mechanical properties of mouse AC: the load-dependent (nonlinear) solid matrix Young's modulus (E) and drained Poisson's ratio (ν). The technique involves confocal microscope-based multiaxial strain mapping of compressed, intact murine AC followed by inverse finite element analysis (iFEA) to determine E and ν. Importantly, this approach yields estimates of E and ν that are independent of the initial guesses used for iterative optimization. As a proof of concept, mechanical properties of AC on the medial femoral condyles of wild-type mice were obtained for both trypsin-treated and control specimens. After proteolytic tissue degradation induced through trypsin treatment, a dramatic decrease in E was observed (compared to controls) at each of the three tested loading conditions. A significant decrease in ν due to trypsin digestion was also detected. These data indicate that the method developed in this study may serve as a valuable tool for comparative studies evaluating factors involved in OA pathogenesis using experimentally induced mouse OA models.