The patellofemoral (PF) joint is a common site for non-specific anterior knee pain. The pathophysiology of patellofemoral pain may be related to abnormal motion of the patella relative to the femur, leading to increased stress at the patellofemoral joint. Patellofemoral motion cannot be accurately measured using conventional motion capture. The aim of this study was to determine the accuracy of a biplane radiography system for measuring in vivo PF motion during walking and stair ascent. Four subjects had three 1.0 mm diameter tantalum beads implanted into the patella. Participants performed three trials each of over ground walking and stair ascent while biplane radiographs were collected at 100 Hz. Patella motion was tracked using radiostereophotogrammetric analysis (RSA) as a "gold standard", and compared to a volumetric CT model-based tracking algorithm that matched digitally reconstructed radiographs to the original biplane radiographs. The average RMS difference between the RSA and model-based tracking was 0.41 mm and 1.97° when there was no obstruction from the contralateral leg. These differences increased by 34% and 40%, respectively, when the patella was at least partially obstructed by the contralateral leg. The average RMS difference in patellofemoral joint space between tracking methods was 0.9 mm or less. Previous validations of biplane radiographic systems have estimated tracking accuracy by moving cadaveric knees through simulated motions. These validations were unable to replicate in vivo kinematics, including patella motion due to muscle activation, and failed to assess the imaging and tracking challenges related to contralateral limb obstruction. By replicating the muscle contraction, movement velocity, joint range of motion, and obstruction of the patella by the contralateral limb, the present study provides a realistic estimate of patellofemoral tracking accuracy for future in vivo studies.