Antiretroviral drug measurements as part of therapeutic drug monitoring are performed in several human immunodeficiency virus (HIV) clinics in developed as well as developing countries. Heparin is routinely used as an anticoagulant for plasma drug estimations. EDTA plasma for drug estimations, if found suitable, would have several practical advantages. In this study, plasma nevirapine (NVP) and efavirenz (EFV) levels were compared in paired blood collected in heparin and EDTA Vacutainer tubes (Greiner Bio-one, Austria). The study involved HIV-infected individuals who were receiving antiretroviral treatment at the Government Hospital of Thoracic Medicine, Tambaram, Chennai, India. Their treatment consisted of administration of NVP or EFV, along with lamivudine and stavudine/zidovudine. Blood was collected at the same time in heparin and EDTA Vacutainer tubes. Plasma concentrations of NVP and EFV were estimated, according to validated methods, by high-performance liquid chromatography (HPLC). The mean NVP concentrations in heparin and EDTA plasma were 8.79 and 8.59 microg/mL, respectively. This difference was not statistically significant (P = 0.203). In the case of EFV, the corresponding values were 3.03 and 2.78 microg/mL, respectively; the difference was significant (P < 0.05). However, the percent variation in EFV between both types of plasma was <10%. Blood collected in heparin or EDTA Vacutainer tubes could be used for plasma NVP determination.