Although antibodies are commercially available to allow investigation into the biology of the age-regulating protein Klotho, problems with antibody specificity and application functionality are significant barriers to progress. Chief among these limitations is the inability of current tools to allow in vivo validation of binding partners originally identified through transfection of tagged proteins. To overcome this barrier, we generated a series of hybridoma cell lines by immunizing rats with a GST-KL1 fusion protein. Purified antibodies generated from these cell lines differentially detect human or mouse Klotho protein via Western blot, immunocyto/histochemistry, and immunoprecipitation. Specificity of antibody binding to Klotho was confirmed by mass spectrometry following immunoprecipitation. With this confidence in antibody specificity, co-immunoprecipitation was utilized to validate the interaction of Klotho/FGFR and Klotho/wnt7a in mouse kidney lysates.