Colloidal nanosphere monolayers—used as a lithography mask for site-controlled material deposition or removal—offer the possibility of cost-effective patterning of large surface areas. In the present study, an automated analysis of scanning electron microscopy (SEM) images is described, which enables the recognition of the individual nanospheres in densely packed monolayers in order to perform a statistical quantification of the sphere size, mask opening size, and sphere-sphere separation distributions. Search algorithms based on Fourier transformation, cross-correlation, multiple-angle intensity profiling, and sphere edge point detection techniques allow for a sphere detection efficiency of at least 99.8%, even in the case of considerable sphere size variations. While the sphere positions and diameters are determined by fitting circles to the spheres edge points, the openings between sphere triples are detected by intensity thresholding. For the analyzed polystyrene sphere monolayers with sphere sizes between 220 and 600 nm and a diameter spread of around 3% coefficients of variation of 6.8–8.1% for the opening size are found. By correlating the mentioned size distributions, it is shown that, in this case, the dominant contribution to the opening size variation stems from nanometer-scale positional variations of the spheres.