In this study we describe how to use a two-dimensional fast Fourier transform (2D FFT) approach to measure fiber alignment in electrospun materials. This image processing function can be coupled with a variety of imaging modalities to assign an objective numerical value to scaffold anisotropy. A data image of an electrospun scaffold is composed of pixels that depict the spatial organization of the constituent fibers. The 2D FFT function converts this spatial information into a mathematically defined frequency domain that maps the rate at which pixel intensities change across the original data image. This output image also contains quantitative information concerning the orientation of objects in a data image. We discuss the theory and practice of using the frequency plot of the 2D FFT function to measure relative scaffold anisotropy and identify the principal axis of fiber orientation. We note that specific degrees of scaffold anisotropy may represent a critical design feature in the fabrication of tissues that will be subjected to well-defined uniaxial mechanical loads. This structural property may also represent a source of guidance cues that can be exploited to regulate cell phenotype.