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A reliable and sensitive HPLC-MS/MS method was firstly developed for the simultaneous determination of hyperoside and astragalin in rat plasma. The plasma sample was prepared by protein precipitation using 300 μL acetonitrile containing 0.1% formic acid. Chromatographic separation was achieved on a C8 column using a gradient elution program with acetonitrile and water containing 0.05% formic acid as the mobile phase. The flow rate was 0.2 mL min−1. The detection was performed in a triple quadrupole tandem mass spectrometer in MRM mode, using negative ionization. The transitions monitored were m/z[M − H] − 463.2 → 300.7 for hyperoside, m/z[M − H] − 447.2 → 283.7 for astragalin and m/z[M − H] − 389 → 226.9 for IS, respectively. The method was linear over the concentration range of 0.5–200 ng mL−1for both analytes. The extraction recovery was above 60%. The intra- and inter-day precision (R.S.D.) values were within 11.04% and accuracy (R.E.) was −3.35% to 10.15% at all QC levels. The method was successfully applied to pre-clinical pharmacokinetic and bioavailability studies of hyperoside and astragalin in rats. Additionally, further research was performed by comparing the differences in the pharmacokinetic behavior between the single substances and the Cuscutaextract after oral or intravenous administration. A lower absolute bioavailability (2.211% for hyperoside and 4.044% for astragalin from Cuscutaextract) and significant differences in the pharmacokinetic behaviors between the Cuscutaextract and single substances were observed in the rat. This method would be beneficial for the pre-clinical research and clinical use of Cuscutaextract.