Background: Gegenqinlian decoction (GQD) is a famous traditional medicine recipe. It is composed of four herbs including Radix Puerariae (GG), Radix Scutellariae (HQ), Rhizoma Coptidis (HL) and Radix Glycyrrhizae (GC), which is widely used for treating gastro-intestinal disorders in the clinical practice of Traditional Chinese medicine (TCM). The aim of this study was to compare the pharmacokinetics of puerarin and daidzein in rats following oral administration of Gegenqinlian Decoction and Radix puerariae extract. Thus, a sensitive and selective liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was developed and validated for simultaneous determination of puerarin and daidzein in rat plasma following oral administration of Gegenqinlian Decoction and Radix Puerariae extract. Materials and Methods: Chromatographic separation was performed on a Shiseido CAPCELL PAK C 18 analytical column (100 mm × 2.0 mm i.d., 5 μm) by linear gradient elution, with water (0.1% formic acid)-acetonitrile (0.1% formic acid) as mobile phase. Detection was carried out by multiple reaction monitoring (MRM) mode using electrospray ionization in the positive ion mode. Results: The calibration curves were linear over a range of 7.80-1560 ng/mL for puerarin and 6.30-1260 ng/mL for daidzein. The intra- and inter-day precision values were less than 13.6% and their average recoveries was in the range of 77.8% and 88.6% for puerarin and was between 76.3 and 86.8% for daidzein, respectively. Conclusion: The validated method was applied to the comparative pharmacokinetic studies of puerarin and daidzein after oral administration of Gegenqinlian Decoction and Radix Puerariae extract. The pharmacokinetic parameters showed that puerarin and daidzein from Gegenqinlian Decoction were absorbed more effectively with slower elimination in rat plasma than that from Radix Puerariae extract. These results revealed that as far as the Radix Puerariae extract was concerned, it is very valuable to be used as a clinical directions of Gegenqinlian Decoction.