Mitochondrial respiratory chain disorders are a group of clinically and genetically heterogeneous diseases. Several mitochondrial (mt)DNA point mutations are responsible for common mitochondrial diseases. These pathogenic mtDNA point mutations are usually heteroplasmic. Molecular diagnosis of the disease requires both qualitative detection of the mutation and quantitative analysis of the mutant heteroplasmy. In this report, two methods based on real-time quantitative polymerase chain reaction (PCR) analysis are used. The first method utilizes wild-type or mutant sequence-specific TaqMan probe, which is labeled with a fluorescent reporter molecule at the 5'-end of the oligonucleotide probe and a quencher at the 3'-end of the probe. The second method utilizes sequence-specific primers to amplify the wild-type or mutant sequence followed by SYBR green detection of PCR products. Both methods allow simultaneous detection and quantification of the mutant mtDNA. In this chapter, we describe the detailed procedures regarding the application of fluorescent probes, and real time quantitative PCR in the molecular diagnosis of mitochondrial DNA disorders.