The efficacy of antiretroviral treatment (ART) may be limited by pharmacological factors. Dose-response relationship exists for many antiretroviral agents, and failure to maintain adequate concentrations may allow the development of viral resistance, thereby decreasing the probability of response of current and subsequent therapies. Fixed dosage of antiretroviral agents may result in different systemic and intracellular concentrations of drugs (interindividual variability). Therapeutic drug monitoring (TDM) consists of individualising dosages with the aim of maximising the efficacy of treatment while minimising its toxicity. The combination of pharmacokinetic-pharmacodynamic relationships for antiretroviral therapy and the presence of a wide interpatient variability in drug exposure support the application of TDM in HIV-infected individuals. Prospective clinical trials assessing the clinical usefulness of this strategy have shown contradictory results, pointing out the need to consider different issues when performing TDM of ART. Pharmacological and resistance data should be combined in one parameter (inhibitory quotient) in antiretroviral-experienced patients. Protease inhibitors exhibit a moderate to high degree of binding to plasma proteins, and in vitro inhibitory concentrations need to be corrected by this factor. The best method to individualize the dosage regimen has not been identified yet. Finally, participation of different laboratories in external cross-validation programmes is crucial to ensure that results are accurate.