We demonstrate the adaption of a further developed Mach-Zehnder interference (MZI) microscope for the rapid 3D characterization of transparent microstructured polymer thin films. In order to quantify the accuracy of the Mach-Zehnder interferometer, comparative film thickness measurements of photolithographically patterned poly(methacrylic acid) polymer brushes are performed employing two alternative techniques: white light profilometry (WIM) and atomic force microscopy (AFM). When the refractive index of the polymer brushes is calculated from MZI data, we obtain a good agreement with results received from an independent method (ellipsometry). In contrast to surface probing techniques such as AFM or WIM. Mach-Zehnder interferometry is a transmitted light method that measures both surface height profiles and refractive index distributions. MZI thus enables the quantification of film homogeneity with respect to height and density variations at the lateral resolution of a refraction limited microscope. We conclude that MZI is an adequate tool for the rapid and non-destructive characterization of structured polymer thin films. This method should be particularly useful for production quality control of microstructured polymer thin films which possess great potential in electronic device fabrication and biotechnology. (C) 2011 Published by Elsevier B.V.