A material testing methodology is investigated in line with non-invasive manner using XRay CT images for characterizing biological tissues. Three-dimensional digital image volumes are generated for initial and loaded states. Tentative displacement field is represented by basis function of B-spline. Unknown parameters included in the functions are identified through an inverse problem minimizing error in terms of pixel intensity between actual deformed image volume and virtual one according to the tentative displacement field of B-spline. A constraint of incompressibility affects well for stable and prompt solution search. The proposed methodology is demonstrated in identification problem of three-dimensional displacement field by using an experimental data of compressive specimen.