Combination of antiretroviral drugs has dramatically improved the prognosis of individuals with HIV infection. However, their long-term benefit is limited by two main factors: the selection of drug-resistant strains and side effects. A large part of the toxicity of antiretroviral drugs has been associated with mitochondrial damage. Nucleoside analogue reverse transcriptase inhibitors (NRTI), which lack the hydroxyl group needed for further DNA chain elongation, block HIV reverse transcriptase. These nucleosides can be mistaken as natural substrates by the polymerase gamma, the enzyme responsible for the replication of mitochondrial DNA (mtDNA). Depletion or damage of mtDNA may affect the aerobic metabolism of carbohydrates and lipids, resulting in the accumulation of pyruvate/lactate and fatty acids, and ultimately in lactic acidosis and lipoatrophy, respectively. However, the relationship between hyperlactatemia and/or lipoatrophy and mtDNA depletion due to NRTIs has not been demonstrated conclusively. The design of methods to measure mtDNA may help to recognize the mitochondrial toxicity of antiretroviral therapy.