To track the pharmacokinetic features of red ginseng (RG), a rapid and sensitive ultra fast liquid chromatographic coupled with electrospray ionization triple quadrupole tandem mass spectrometry (UFLC–MS/MS) method was developed for simultaneous quantification of twenty-one ginsenosides and their three aglycones, including 18 prototype compounds (ginsenosides Rb1, Rb2, Rc, Rd, Re, Rg1, Rg5, Rh4, Rk1, Rk3, 20(S)-Rf, 20(S)-Rg2, 20(R)-Rg2, 20(S)-Rg3, 20(R)-Rg3, 20(S)-Rh1, 20(R)-Rh1, 20(S)-NG-R2), and 6 metabolites (ginsenosides 20(S)-Rh2 and Rh3, 20(S)-protopanaxadiol (PPD), 20(S)-protopanaxatriol (PPT), 20(R)-PPT, ginseng saponin compound K) of RG in rat plasma after oral administration of RG water extract at a single dose of 4 g/kg body weight to rats. All analytes with internal standard (digoxin) were detected by multiple reaction monitoring in negative ionization mode and separated on an ACQUITY UPLC® BEH RP-C18 column (1.7 μm, 100 × 2.1 mm). This established method was well validated in terms of linearity, sensitivity, intra- and inter-day precisions, accuracy, recovery, matrix effect, stability, and had a lower limit of quantification at the concentration range of 0.12–8.12 ng/mL for all of analytes. This UFLC–MS/MS approach was successfully applied to the pharmacokinetic study for RG water extract in rats. We firstly proposed that Rb1, Rb2, Rc, Rd, Rg1, Rg5, 20(S)-Rg3, 20(S)-Rh2, and 20(S)-PPD measured in rat plasma were suitable pharmacokinetic markers of RG extract in rats due to their high systemic exposure levels. Thus, this specific and reliable method will be useful for future applications to pharmacokinetic studies for various sources of ginsenoside samples and Panax herbs in vivo.