Systemic autoimmune diseases are characterized by the presence in patient sera of high titers of autoantibodies directed against self-antigens. The characterization of specific autoantigen-autoantibody system is very important for research as well as for diagnostic tools and several techniques can be employed in such immunological studies. In this review an overview of the different biochemical methods of antigen preparation and the specific methodological applications is given, also describing potential critical aspects and benefits of the different protocols.