An in vitro acoustic microscopy method for the quantitative characterization of biological hard tissues at a microscopic scale is described. The acoustic impedance is measured as a tissue parameter, which is closely related to its elasto-mechanical properties. Contrast influences caused by defocus, edges and surface inclinations, respectively are either compensated or excluded from the measurement using the Multi Layer Analysis technique for data acquisition and evaluation. A raster grid was used to validate the capabilities and limitations of the method and results obtained from human cortical bone are shown. The comparison of different evaluation methods demonstrate the significance of a sophisticated analysis under consideration of topographical and system parameters. Cortical bone impedance maps showed a strong dependence on the anatomical structures and the mean values were found to be in the range from 3.5 to 6.5 Mrayl within one single osteon.