Isocitrate dehydrogenase 2 (IDH2) mutations have been reported in gliomas, osteosarcomas, cartilaginous tumors, giant cell tumors of bone, and acute myeloid leukemias. Although IDH2 catalyzes the oxidative carboxylation of isocitrate to α-ketoglutarate (α-KG) in mitochondria, mutated IDH2 proteins possess the ability to change α-KG into the oncometabolite R(-)-2-hydroxyglutarate (2-HG). To date, several monoclonal antibodies (mAbs) specific for IDH2 mutations have been established, such as KMab-1 against IDH2-R172K, MMab-1 against IDH2-R172M, and WMab-1 against IDH2-R172W. Although a multi-specific mAb MsMab-1 reacted with IDH2-R172G and IDH2-R172S, a mono-specific mAb against IDH2-R172S has not been established. In this study, we established a novel mAb SMab-2, which recognizes IDH2-R172S but not with wild type IDH2 in ELISA. Although SMab-2 reacted with both IDH1-R132S and IDH2-R172S expressed in Escherichia coli, it reacted with only IDH2-R172S expressed in U-2 OS osteosarcoma cells. Furthermore, SMab-2 recognized endogenous IDH2-R172S protein expressed in SW1353 chondrosarcoma cells in Western blot and immunocytochemical analyses. SMab-2 is expected to be useful for diagnosis of IDH2-R172S-bearing tumors.