Assessment of the mutational status of the isocitrate dehydrogenase 1/2 (IDH1/2) gene has become an integral part of the standard diagnostic procedure and, therefore, needs to be accurate. This may, however, be compromised by various factors including the method of analysis and a low tumor cell content. We have developed a rapid, sensitive and robust assay to detect all types of mutation in either IDH1 or IDH2 using pyrosequencing. The efficacy of detecting mutation was evaluated using a panel of control plasmids representing all the different types of IDH1/2 mutation and a set of 160 tumor specimens. The sensitivity of the assays was examined by a serial dilution analysis performed on samples containing various ratios of wild-type and mutant alleles. The pyrosequencing assay detected as little as 5 % of mutant alleles for most mutation types, while conventional Sanger sequencing required the presence of at least 20 % of mutant alleles for identifying mutations. The pyrosequencing assay detected IDH1/2 mutations in three samples which were missed by Sanger sequencing due to their low tumor cell contents. Our assay is particularly useful for the analysis of a large number of specimens as in a retrospective clinical study for example.