The in vivo assessment of the quality of cartilage is a critical tool to determine how to clinically treat damaged cartilage. Utilizing nondestructive indentation, a hand-held instrument (ACTAEON Probe) was used to rapidly measure the stiffness of articular cartilage, which can then be correlated to cartilage condition. An integrated series of tests were performed to determine whether the Probe is sensitive enough to detect early degenerative changes in articular cartilage and to examine if it is dependent on the thickness of the cartilage being tested. Results demonstrated that there is no effect of thickness on the Probe measurements for clinically relevant human cartilage samples, that the Probe is sensitive enough to detect different levels of cartilage degeneration, and that Probe data correlate well with traditional creep indentation testing. This study demonstrates the utility of the Probe for detecting degenerative changes in animal and human joints.