The use of biplanar videoradiography technology has become increasingly popular for evaluating joint function in vivo. Two fundamentally different methods are currently employed to reconstruct 3D bone motions captured using this technology. Marker-based tracking requires at least three radio-opaque markers to be implanted in the bone of interest. Markerless tracking makes use of algorithms designed to match 3D bone shapes to biplanar videoradiography data. In order to reliably quantify in vivo bone motion, the systematic error of these tracking techniques should be evaluated. Herein, we present new markerless tracking software that makes use of modern GPU technology, describe a versatile method for quantifying the systematic error of a biplanar videoradiography motion capture system using independent gold standard instrumentation, and evaluate the systematic error of the W.M. Keck XROMM Facility’s biplanar videoradiography system using both marker-based and markerless tracking algorithms under static and dynamic motion conditions. A polycarbonate flag embedded with 12 radio-opaque markers was used to evaluate the systematic error of the marker-based tracking algorithm. Three human cadaveric bones (distal femur, distal radius, and distal ulna) were used to evaluate the systematic error of the markerless tracking algorithm. The systematic error was evaluated by comparing motions to independent gold standard instrumentation. Static motions were compared to high accuracy linear and rotary stages while dynamic motions were compared to a high accuracy angular displacement transducer. Marker-based tracking was shown to effectively track motion to within 0.1 mm and 0.1 deg under static and dynamic conditions. Furthermore, the presented results indicate that markerless tracking can be used to effectively track rapid bone motions to within 0.15 deg for the distal aspects of the femur, radius, and ulna. Both marker-based and markerless tracking techniques were in excellent agreement with the gold standard instrumentation for both static and dynamic testing protocols. Future research will employ these techniques to quantify in vivo joint motion for high-speed upper and lower extremity impacts such as jumping, landing, and hammering.