Red blood cell (RBC) alloimmunization and antibodies formation against non-self antigens on red cells may occur after blood transfusion, pregnancies or other exposures. The RBC alloimmunization rate varies from 2% to 6% according to recent studies. The antibody screen is performed to identify or confirm the presence of antibodies in patient’s serum or plasma, as a preoperative or pretransfusion test. The antibody identification process and major crossmatch are critical steps of risk management in transfusion medicine. The aim of this article is to describe a flow chart of the antibody identification. I report three educational examples of case studies associated with the negative direct antiglobulin test and clinically significant single and multiple alloantibodies using the gel method, Anti-M, Anti-c and Anti-E, Anti-Jka and Anti-s. Furthermore, I provide a critical analysis of the current literature on the topic. The flow chart of the antibody identification may simplify the process and possibly reduce errors in routine workflow.