Functions of cells and their dynamic behavior are influenced by the Young's modulus of the underlying substrate (e.g. differentiation of stem cells into different lineages or migration of fibroblasts). The defined influence of cellular functions is dependent on a reliable determination of the Young's modulus. The Atomic Force Microscopy (AFM) can determine the Young's modulus on the cellular scale. Nonetheless, the obtained values of Young's moduli published by different authors vary even though similar experiments are carried out. Main reasons are insufficient transparency of experimental parameters and little discussions about crucial factors that might alter the determined Young's moduli. Here, results of indentation experiments carried out with the AFM are described and discussed. Cantilevers were pressed on soft polymer samples (Polyacrylamide, PAM, and Polydimethylsiloxane, PDMS) with different setpoint-forces and indentation speeds. PAM and PDMS are commonly used polymers for biological substrates due to their easily tuned physiological relevant Young's moduli. In the presented work, the Young's moduli were calculated from a Hertzian fit to the extend curve of recorded force-distance curves. The huge distribution of calculated Young's moduli shows a necessity for elaborate discussions about measurement and analyzing parameters. The quality of the fit, the applied setpoint-force as well as a strong position dependency were found to be major aspects, whereas variations of the Young's moduli within one position are negligible small. A Matlab program was developed for mainly automatic analyses of force-distance curves and calculations of Young's moduli. A major advantage of the homemade program is the display of so called E-d curves which enables a systematic strategy to find sufficient high setpoint-forces to reach stable Young's moduli. Altogether, the presented work proposes a new procedure for indentation experiments for the mechanical characterization of biological substrates.