Janus kinases (JAKs) are tyrosine kinases called JAK-1, JAK-2, JAK-3, and Tyk-2, which have been shown to participate in the signaling pathways of several cytokines that are believed to play a key role in several autoimmune-mediated disorders including rheumatoid arthritis (RA). However, the availability of JAK-specific antibodies to be used in investigative efficacy studies in RA models is very limited. Therefore, in this study we developed and characterized a JAK-2-specific antibody that was used to evaluate its immunohistochemical expression in the joints of a rat adjuvant-induced arthritis (rAIA) RA preclinical model. An immunogen peptide sequence design was used to generate JAK-2-specific mouse, rat, and human polyclonal antibodies. JAK-2 plasmid cDNA was then generated and HEK293 transfected cell lines, gel electrophoresis, immunoblotting, and immunohistochemistry were used to further characterize the generated JAK-2 antibodies. We show that the generated JAK-2 antibody exhibits specificity and lacks cross-reactivity to JAK-1 and JAK-3. In addition, marked JAK-2 expression is shown in the rAIA in mixed inflammatory cells (macrophages and neutrophils), mast cells, and bone marrow elements. In conclusion, we show the development and characterization of a JAK-2-specific antibody that can be used in investigative and mechanistic studies such as preclinical efficacy models.