Detecting and quantifying generalized mitochondrial heteroplasmy is essential if the field of mitochondrial genetics is to advance in the arena of complex genetic disorders. The majority of techniques used to detect and quantify mitochondrial heteroplasmy focus on a known mutation or polymorphism. The necessity of knowing the mitochondrial DNA (mtDNA) change beforehand means that non-specific heteroplasmy in general cannot be assessed. In this study, we assessed the extent that denaturing high-performance liquid chromatography (dHPLC) could detect and quantify mitochondrial heteroplasmy from cerebrospinal fluid (CSF). Although we used a known polymorphism to assess reliability and sensitivity of this technique, a distinct advantage to using dHPLC for heteroplasmy detection is that the entire fragment is screened for variability and any unique fragments will be detected regardless of the placement or type of change. Our results demonstrate that dHPLC can consistently and reliably detect mitochondrial heteroplasmy in a CSF sample down to 0.01%. In addition, the level of heteroplasmy was consistent with peak height for each homoduplex, giving a reliable method to quantify level of heteroplasmy.