Monoclonal antibody technology has generated invaluable tools for both the analytical and clinical sciences. However, standard immunization approaches frequently fail to provide monoclonal antibodies with the desired specificity. Subtractive immunization provides a powerful alternative to standard immunization and allows for the production of truly unique antibodies. With the intent of targeting specific epitopes within the proteome, subtractive immunization has been broadly and successfully implemented for the production of monoclonal antibodies otherwise unobtainable by standard immunization. Subtractive immunization utilizes a distinct immune tolerization approach that can substantially enhance the generation of monoclonal antibodies to desired antigens. The approach is based on tolerizing the host animal to immunodominant or otherwise undesired antigen(s) (tolerogen) that may be structurally or functionally related to the antigen of interest. Tolerization of the host animal can be achieved through one of three methods: High Zone, Neonatal, or Drug-induced tolerization. The tolerized animal is then inoculated with the desired antigen (immunogen) and antibodies generated by the subsequent immune response are screened for the desired antigenic reactivity. Over the past 15 years a large number of investigators have used the subtractive approach with cleverly chosen tolerogen-immunogen combinations and successfully generated uniquely reactive antibodies which are often neutralizing or function-blocking. This review will focus on the implementation of subtractive immunization for the production of antibodies otherwise unobtainable by standard immunization.