In a previous paper, we reported the virtual axis finder, which is a new method for finding the rotational axes of the knee. The virtual axis finder was validated through simulations that were subject to limitations. Hence, the objective of the present study was to perform a mechanical validation with two measurement modalities: 3D video-based motion analysis and marker-based roentgen stereophotogrammetric analysis (RSA). A two rotational axis mechanism was developed, which simulated internal-external (or longitudinal) and flexion-extension (FE) rotations. The actual axes of rotation were known with respect to motion analysis and RSA markers within and and and , respectively. The orientation and position root mean squared errors for identifying the longitudinal rotation (LR) and FE axes with video-based motion analysis (0.26 deg, 0.28 m, 0.36 deg, and 0.25 mm, respectively) were smaller than with RSA (1.04 deg, 0.84 mm, 0.82 deg, and 0.32 mm, respectively). The random error or precision in the orientation and position was significantly better ( and , respectively) in identifying the LR axis with video-based motion analysis (0.23 deg and 0.24 mm) than with RSA (0.95 deg and 0.76 mm). There was no significant difference in the bias errors between measurement modalities. In comparing the mechanical validations to virtual validations, the virtual validations produced comparable errors to those of the mechanical validation. The only significant difference between the errors of the mechanical and virtual validations was the precision in the position of the LR axis while simulating video-based motion analysis (0.24 mm and 0.78 mm, ). These results indicate that video-based motion analysis with the equipment used in this study is the superior measurement modality for use with the virtual axis finder but both measurement modalities produce satisfactory results. The lack of significant differences between validation techniques suggests that the virtual sensitivity analysis previously performed was appropriately modeled. Thus, the virtual axis finder can be applied with a thorough understanding of its errors in a variety of test conditions.