An improved HPLC fluorimetric method for the quantification of enfuvirtide in plasma of HIV-infected subjects was described and validated. The use of an internal standard improved the reproducibility and precision of the analysis. Our method showed lower limits of detection and quantification (LOD = 32 ng/mL, LOQ = 78 ng/mL), lower intraday (RSD% 1.25-2.95) and interday (RSD% 1.75-4.69) coefficients of variation, greater recovery (>100%), lower duration (16 minutes) and lower cost than previously described fluorimetric methods. Therefore, this method can be used as a reliable tool for pharmacokinetic studies of enfuvirtide.