The in vivo evaluation of a material to be used in clinics is a long trial and requires a well planned sequence of steps. In vivo tests are complementary to those performed in vitro that provide necessary and useful results to be added to those found in the in vivo testing, and strongly reduce the number of animals used. Animal studies require first of all the compliance to the ethical and legal rules on animal experimentation. The procedures are standardized at an international level. In particular the rules of ISO 10993 are the guidelines for these investigations. In the procedure of validation of a material, the choice of more than one animal species for the performing of the tests, namely a small/medium and a large size animal should be appropriate. Considering the research on biomaterials, the use of small animals can be acceptable in the early stage of testing, but for the evaluation of prototypes biofunctionality large animals should better approximate the human environment. According to the European and International rules, the experiments must be performed in authorized laboratories, and surgical interventions in well equipped operatory rooms and in general anesthesia. Clinical monitoring and X ray investigations allow the scheduled follow-up on living animals. After the sacrifice histology and histomorphometry provide useful information about the interactions between the material and the tissues, together with biomechanical test. More recently computerized microtomography, a non destructive procedure, is added to the methods of study on laboratory animals allowing high resolution images and the visualization of the internal structures of the objects.