Many tumors, especially gliomas, contain an isocitrate dehydrogenase (IDH) mutation that can be used for clinical diagnosis and prognosis. Our study aimed to develop a new reliable detection assay for IDH1 and IDH2 mutations for clinical diagnosis based on the allele-specific (AS) coamplification with lower denaturing-polymerase chain reaction (COLD-PCR) and probe melting curve analysis (PMCA). The method includes 3 elements allowing for the sensitive detection of low-abundance mutations: (1) PCR amplification of the target fragments with AS primers; (2) COLD-PCR; and (3) PMCA for differentiating the different mutations after amplification. We conducted a blinded study with 45 paraffin-embedded gliomas specimens and 13 fresh specimens screened for IDH mutations using Sanger sequencing. Concordance between the results of our AS-COLD-PCR/PMCA assay and Sanger sequencing was 100%. Our assay appeared to be superior to direct sequencing with a much higher sensitivity of 0.4% mutations. In summary, our assay is a cost-effective, convenient, and sensitive method for detecting IDH mutations and could be applied in the clinical setting to assess small brain biopsies.