Alamifovir disoproxil fumarate (ADF) is a novel ester prodrug of alamifovir, which is currently as a promising antiviral candidate under investigation. This paper is aimed to develop rapid, sensitive and specific LC-MS/MS methods for the quantification of ADF and its active metabolite 602076 in rat plasma. According to the significantly different chemical properties of the compounds, two sets of liquid chromatography and ionization modes were used for determining the concentration of ADF and 602076 in rat plasma, separately. Following liquid-liquid extraction with n-hexane:dichloromethane:isopropanol (100:50:5, v/v/v), the ADF and internal standard (gliclazide) were separated on a Phenomenex Gemini C(18) column (150 mm × 2.0 mm, 5 µm) with a mobile phase consisting of methanol:water:formic acid (70:30:0.1, v/v/v). A tandem mass spectrometer equipped with electrospray ionization (ESI) source was used as the detector and operated in positive ion mode. The metabolite 602076 and the internal standard ZHY81018 were extracted from plasma by protein precipitation with acetonitrile. Chromatographic separation was performed on a Capcell MG C(18) column (150 mm × 2.0 mm, 5 µm) with a mobile phase consisting of acetonitrile and water (20:80, v/v). The MS/MS detection was operated in negative ion mode using an ESI source. The linear concentration ranges of the calibration curves were 2.5-500 ng/mL for ADF and 2.5-1000 ng/mL for 602076. The intra-assay RSD for quality control (QC) samples were from 3.3% to 6.7% for ADF, and 4.0% to 6.1% for 602076. The inter-assay RSD for QC samples were from 4.9% to 14.7% for ADF, and 2.6% to 4.4% for 602076. The relative errors for QC samples were from -10.6% to 1.9% for ADF, and 0.2% to 2.6% for 602076. The methods were successfully applied in the investigation of the pharmacokinetic profile of ADF, alamifovir and 602076 in rats. The results showed that ADF was rapidly metabolized to its active metabolite 602076 after oral absorption, with no detectable unchanged drug. The oral bioavailability of ADF was about 3 times higher than that of alamifovir.