Even though ultrasound imaging is widely used in clinical diagnosis and image-guided interventions, the field is far behind other areas of clinical image analysis, such as MRI, CT and X-ray mammography. In this book, non-destructive and non-invasive ultrasound characterization techniques were developed to study the tissue micro-structural details using high frequency spectral ultrasound imaging (SUSI). Spectrum of the backscattered RF ultrasound data contains tissue microstructural properties such as scatterer density, size and concentration and can be estimated with an inverse model. These techniques were explored in in-vitro conditions of acellular and cellular tissue engineered constructs and then on ex-vivo cardiac tissues for their micro-structural characterization. Even though the results from the developed techniques show great promise in in-vitro and ex-vivo settings, additional work needs to be carried out to demonstrate the applicability of the techniques in in-vivo, particularly to translate these techniques into clinic.