Aim: To study the pharmacokinetics of sifuvirtide, a novel anti-human immunodeficiency virus (HIV) peptide, in monkeys and to compare the inhibitory concentrations of sifuvirtide and enfuvirtide on HIV-1-infected-cell fusion. Methods: Monkeys received 1.2 mg/kg iv or sc of sifuvirtide. An on-line solid-phase extraction procedure combined with liquid chromatography tandem mass spectrometry (SPELC/MS/MS) was established and applied to determine the concentration of sifuvirtide in monkey plasma. A four-~(127)I iodinated peptide was used as an internal standard. Fifty percent inhibitory concentration (IC_(50)) of sifuvirtide on cell fusion was determined by co-cultivation assay. Results: The assay was validated with good precision and accuracy. The calibration curve for sifuvirtide in plasma was linear over a range of 4.88-5000μg/L, with correlation coefficients above 0.9923. After iv or sc administration, the observed peak concentrations of sifuvirtide were 10 626±2 886μg/L and 528±191μg/L, and the terminal imination half-lives (T_(1/2)) were 6.3±0.9 h and 5.5±1.0 h, respectively. After sc, T_(max) was 0.25-2h, and the absolute bioavailability was 49%±13%. Sifuvirtide inhibited the syncytium formation between HIV-1 chronically infected cells and uninfected cells with an IC_(50) of 0.33μg/L. Conclusion: An on-line SPE-LC/MS/MS approach was established for peptide pharmacokinetic studies. Sifuvirtide was rapidly absorbed subcutaneously into the blood circulation. The T_(1/2) of sifuvirtide was remarkably longer than that of its analog, enfuvirtide, reported in healthy monkeys and it conferred a long-term plasma concentration level which was higher than its IC_(50) in vitro. Aim: To study the pharmacokinetics of sifuvirtide, a novel anti-human immunodeficiency virus (HIV) peptide, in monkeys and to compare the inhibitory concentrations of sifuvirtide and enfuvirtide on HIV-1-infected-cell fusion. Methods: Monkeys received 1.2 mg / kg iv or sc of sifuvirtide. An on-line solid-phase extraction procedure combined with liquid chromatography tandem mass spectrometry (SPELC / MS / MS) was established and applied to determine the concentration of sifuvirtide in monkey plasma. Fifty percent inhibitory concentration (IC_ (50)) of sifuvirtide on cell fusion was determined by co-cultivation assay. Results: The assay was validated with good precision and accuracy. The calibration curve for sifuvirtide in plasma was linear over a range of 4.88-5000 μg / L with correlation coefficients above 0.9923. After iv or sc administration, the observed peak concentrations of sifuvirtide were 10 626 ± 2 886 μg / L and 528 ± 19 1 μg / L, and the terminal imination half-lives (T_ (1/2)) were 6.3 ± 0.9 h and 5.5 ± 1.0 h, respectively. After sc, T_max was 0.25-2h, and the absolute bioavailability was 49 % ± 13%. Sifuvirtide inhibited the syncytium formation between HIV-1 chronically infected cells and uninfected cells with an IC 50 (50) of 0.33 μg / L. Conclusion: An on-line SPE-LC / MS / MS approach was established for peptide pharmacokinetic studies. Sifuvirtide was rapidly absorbed subcutaneously into the blood circulation. The T_ (1/2) of sifuvirtide was remarkably longer than that of its analog, enfuvirtide, reported in healthy monkeys and it conferred a long-term plasma concentration level which was higher than its IC_ (50) in vitro.