Abstract A pharmacokinetic study based on single oral administration of Tenofovir disoproxil fumarate 300 mg (tablets) to Argentinean male volunteers under fasting conditions is presented. The obtained values for test and reference products were 238±62 and 261±73 ng mL-1 for Cmax; 1658±466 and 1801±529 ng mL-1 h for AUC0–48h; 1845±579 and 2015±639 ng mL-1 h for AUC0–∞, respectively. The 90% confidence intervals obtained by analysis of variance were 85.0-97.2% for ln-Cmax, 87.2-97.9% for ln-AUC0–48h and 85.5-98.1% for ln-AUC0–∞, which are within the acceptance range of 80-125%. Both products were bioequivalent in terms of rate and extent of drug absorption and therefore interchangeable. The analytical methodology was optimized and validated for determination of Emtricitabine (FTC), Lamivudine (3TC) and Tenofovir (TFV) in human plasma samples; which is the pharmaceutical combination most commonly used for treating HIV-infected patients. The analytical methodology was based on solid phase extraction (SPE) using Oasis® MCX mixed-mode cartridges coupled to Liquid Chromatography and electrospray tandem mass spectrometry (LC-ESI-MS/MS). The analytical methodology was validated according to the US Food and Drug Administration guidelines. Under optimized conditions, the analytical methodology lead to work within a clinical of 11-5434, 11-5452 and 13-397 ng mL-1 for FTC, 3TC and TFV, respectively; resulting wider than others previously reported. The intra and inter batch precision (%RSD) across three validation runs was less than 14%. The accuracy determined at four QC levels (LOQ, Low, Middle and High) was within 13%, in terms of percent relative error.