Combining accurate bone kinematics data from biplane radiography with cartilage models from magnetic resonance imaging, it is possible to estimate tibiofemoral cartilage contact area and centroid location. Proper validation of such estimates, however, has not been performed under loading conditions approximating functional tasks, such as gait, squatting, and stair descent. The goal of this study was to perform an in vitro validation to resolve the accuracy of cartilage contact estimations in comparison to a laser scanning gold standard. Results demonstrated acceptable reliability and accuracy for both contact area and centroid location estimates. Root mean square errors in contact area averaged 8.4% and 4.4% of the medial and lateral compartmental areas, respectively. Modified Sorensen-Dice agreement scores of contact regions averaged 0.81 ± 0.07 for medial and 0.83 ± 0.07 for lateral compartments. These validated methods have applications for in vivo assessment of a variety of patient populations and physical activities, and may lead to greater understanding of the relationships between knee cartilage function, effects of joint injury and treatment, and the development of osteoarthritis.