We propose a simple method able to quantify three-dimensional (3D) anisotropy in topographic microstructure measurements. The anisotropy quantification of 3D surface data is based on a horizontal-cut analysis yielding level sets. For a specific level set, we study xy anisotropy with the use of a directional chord length analysis. In the height profile direction, the z direction, anisotropy is quantified using the framework of mean Euler characteristic and more generally that of Minkowski functionals. We exemplify the method with the use of 3D heights profiles from atomic force microscopy measurements. Copyright © 2012 John Wiley & Sons, Ltd.