Fluoroscopic techniques have been recently used to detect in vivo knee joint kinematics. This article presents a technique that uses two fluoroscopes to form a dual orthogonal fluoroscopic system for accurately measuring in vivo 6DOF total knee arthoplasty (TKA) kinematics. The system was rigorously validated and used to investigate in vivo kinematics of 12 patients after cruciate-retaining TKA. In a repeatability study, the pose of two different TKA components was reproduced with standard deviations (SD) of 0.17 mm and 0.57° about all three axes. In an accuracy study, the reproduced component positions were compared to the known component positions. Position and rotation mean errors were all within 0.11 mm and 0.24°, with SD within 0.11 mm and 0.48°, respectively. The results of this study show that the matching process of the imaging system is able to accurately reproduce the spatial positions and orientations of both the femoral and tibial components. For CR TKA patients, a consistent anterior femoral translation was observed with flexion through 45° of flexion, and thereafter, the femur translated posteriorly with further flexion. The medial-lateral translation was measured to be less than 2 mm throughout the entire flexion range. Internal tibial rotation steadily increased through maximum flexion by approximately 6°. Varus rotation was also measured with flexion but had a mean magnitude less than 2.0°. In conclusion, the dual orthogonal fluoroscopic system accurately detects TKA kinematics and is applicable towards other joints of the musculoskeletal system, including the wrist, elbow, shoulder, ankle, and spine.