The behavior of cells and tissue is greatly influenced by the mechanical properties of their environment. For studies on the interactions between cells and soft matrices, especially those applying traction force microscopy the characterization of the mechanical properties of thin substrate layers is essential. Various techniques to measure the elastic modulus are available. Methods to accurately measure the Poisson ratio of such substrates are rare and often imply either a combination of multiple techniques or additional equipment which is not needed for the actual biological studies. Here we describe a novel technique to measure both parameters, the Youngs's modulus and the Poisson ratio in a single experiment. The technique requires only a standard inverted epifluorescence microscope. As a model system, we chose cross-linked polyacrylamide and poly-N-isopropylacrylamide hydrogels which are known to obey Hooke's law. We place millimeter-sized steel spheres on the substrates which indent the surface. The data are evaluated using a previously published model which takes finite thickness effects of the substrate layer into account. We demonstrate experimentally for the first time that the application of the model allows the simultaneous determination of both the Young's modulus and the Poisson ratio. Since the method is easy to adapt and comes without the need of special equipment, we envision the technique to become a standard tool for the characterization of substrates for a wide range of investigations of cell and tissue behavior in various mechanical environments as well as other samples, including biological materials.