A new method has been developed to define the directional parameter and characterize the structural anisotropy of a highly porous structure with extensive pore interconnectivity and surface area, such as scaffolds in tissue engineering. This new method called intercept segment deviation (ISD) was validated through the comparison of structural anisotropy from ISD measurements with mechanical anisotropy from finite-element stress analysis. This was carried out on a generated two-dimensional (2D) image of a two-phase material and a real three-dimensional (3D) image of a tissue scaffold. The effect of tissue regeneration and scaffold degradation on the anisotropy of the scaffold was discussed. The performance of other methods for quantification of the directional parameter was also assessed. The results indicate that the structural anisotropy obtained from this new method conforms to the actual mechanical anisotropy and provides a better prediction of the material orientation than the other methods for the 2D and 3D images studied.