Ginsenoside Re (G-Re) improved the memory function of experimental animals in a preclinical study. Several types of saponins including G-Rg1, G-Rg2, G-F1, G-Rh1, and protopanaxatriol (PPT) may be the metabolites of G-Re according to reports from preclinical trials. In order to support a study of the pharmacokinetics of G-Re, an analytical method for G-Re and the co-detection of its probable metabolites using liquid chromatography tandem mass spectrometry (LC–MS/MS) was developed and validated. Solid phase extraction was utilized in the sample preparation. Separation of the analytes was achieved using a gradient elution (0.05% formic acid–methanol–acetonitrile, each organic phase containing 0.05% formic acid) at a flow rate of 0.3 mL/min with a retention time of approximately 2.88 min for G-Re. Data were acquired in the multiple reaction mode (MRM) and the linear range of the standard curve of plasma and urine samples for G-Re was 0.05–20 ng/mL with r2 ≥ 0.99. In the analysis of probable metabolites, G-Re, G-Rg1, G-F1, G-Rh1 and PPT were all detected in samples; however, G-Rg2 was not detected.