This chapter starts with an introduction of the issues related to biomaterials characterization and the overall strategies of performing three levels of tests: initial tests for defining the general properties of a biomaterial and for quality assurance; level II tests for defining the properties closely related to the end-use applications including testing of retrievedmaterials afterin vitro or in vivo exposure to the host biological environment; level III tests for evaluating the biological responses of blood or tissue to the implanted biomaterial. The methods for biomaterial bulk property analysis are then reviewed with discussions focused on two new technologies: X-ray micro-computed tomography(μCT) and X-ray microdiffraction. The rest of this chapter provides detailed reviews on selected surface analysis techniques including microscopy methods: transmission electron microscopy (TEM), scanning electron microscopy (SEM), scanning tunneling microscopy (STM), atomic force microscopy (AFM), confocal laser scanning microscopy (CLSM); spectroscopy methods: X-ray photoelectron spectroscopy (XPS), second ion mass spectrometry (SIMS), Infrared spectroscopy; microspectroscopy and spectral imaging methods: IR microspectroscopic imaging, Raman microspectroscopic imaging; thermodynamic methods: contact angle analysis; and emerging optical methods for in vivo analysis: rapid Raman spectroscopy, multi-photon excitation (MPE) microscopy, optical coherence tomography (OCT). The chapter ends by emphasizing the importance of combing multiple analysis methods for a comprehensive solution of biomaterial characterization for specific applications.