Expansion microscopy evolved as an interesting alternative in the field of super-resolution microscopy by physically expanding the target sample to increase its size by a multiple and to subsequently image the sample by conventional fluorescence microcopy. But the quantitative determination of the expansion factor remains challenging, since microscopic measurement standards are missing. Here we use DNA nanorulers in combination with expansion microscopy to expand the fluorescently marked rulers in 2D and to determine the intermark distances before and after expansion. We compare macroscopic and microscopic expansion factors where we found substantial deviations, which should be considered for the quantitative discussion of expansion microscopy data of biological cells and tissues. The demonstrated proof-of-principle opens a new field with the potential to answer questions with respect to quantitation of microscopic magnification and the introduced expansion nanorulers present a powerful tool to reveal the true origin of anisotropy and heterogeneity in expanded microscopy specimen.