Background: Numerous studies have demonstrated significant asymmetries in unilateral amputee gait. The underlying dissimilarities between prosthetic and intact limbs have not yet been widely examined. To gain more insight into the functionality of asymmetries, we propose a new tool, the symmetry function (SF), to evaluate the symmetry of walking in terms of kinematic and dynamic variables of patients after unilateral transfemoral amputation and to identify areas with the largest side deviations in the movement cycle. Methods: An instrumented motion analysis system was used to register the gait of fourteen patients after unilateral trans-femoral amputation (TFA). Measurements involved evaluating the time series of gait variables characterizing a range of motion and the time series of the ground reaction force components. Comparison of the involved limb with the uninvolved limb in TFA patients was carried out on the basis of the SF values.Results: The symmetry function proved to be an excellent tool to localize the regions of asymmetry and their positive or negative directions in the full gait cycle. The difference between sides revealed by the symmetry function was the highest for the pelvis and the hip. In the sagittal plane, the pelvis was asymmetrically tilted, reaching the highest SF value of more than 25% at 60% cycle time. In the transverse plane, the pelvis was even more asymmetrically positioned throughout the entire gait cycle (50% difference on average). The hip in the frontal plane reached a 60% difference in SF throughout the single support phase for the prosthetic and then for the intact limb. Conclusions: The symmetry function allows for the detection of gait asymmetries and shifts in the center of gravity and may assess the precise in time adaptation of prostheses and rehabilitation monitoring, especially in unilateral impairments.Trial registration: The trial registration number (TRN): 379991 issued by the Australian New Zealand Clinical Trials Registry (ANZCTR) on 07.05.2020 (retrospectively registered).