A method for automated quantification of the alignment of one-dimensional (1D) nanostructures from microscopy imaging is presented. Nanostructure alignment metrics are formulated and shown to be able to rigorously quantify the orientational order of nanostructures within a two-dimensional domain (surface). A complementary image processing method is also presented which enables robust processing of microscopy images where overlapping nanostructures might be present. Scanning electron microscopy (SEM) images of nanowire-covered surfaces are analyzed using the presented methods and it is shown that past single parameter alignment metrics are insufficient for highly aligned domains. Through the use of multiple parameter alignment metrics, automated quantitative analysis of SEM images is shown to be possible and the alignment characteristics of different samples are able to be quantitatively compared using a similarity metric. The results of this work provide researchers in nanoscience and nanotechnology with a rigorous method for the determination of structure/property relationships, where alignment of 1D nanostructures is significant.