A new multiple columns HPLC method for simultaneous determination of 16 characteristic components, 5 nucleobases and nucleosides (uracil, cytidine, uridine, guanosine and adenosine), and 11 saponins (notoginsenoside R1, ginsenoside Rg1, ginsenoside Re, notoginsenoside R4, notoginsenoside Fa, ginsenoside Rb1, notoginsenoside R2, ginsenoside Rg2, ginsenoside Rh1, ginsenoside Rd and notoginsenoside K), in the root of Panax notoginseng, a valued traditional Chinese medicinal herb, were developed. Notoginsenoside R4, Fa and K were first quantitatively determined in P. notoginseng. The 5 nucleobases and nucleosides compounds were separated on a Zorbax SB-Aq column (150 × 4.6 mm, 5.0 μm) and 11 saponins were analyzed using a Zorbax Bonus-RP column (150 × 4.6 mm, 5.0 μm) with column switching. The column temperature was set at 30 °C. Mobile phase was composed of 5 mM ammonium acetate aqueous (A), water (B) and acetonitrile (C) using a gradient elution. The flow rate was 1.5 mL/min and detection wavelengths were set at 260 nm for nucleobases and nucleosides, and 203 nm for saponins. The developed method had good repeatability and sensitivity for quantification of 16 analytes with overall precision (including intra- and inter-day) less than 3% (RSD), and LOD and LOQ were less than 1.33 μg/mL and 5.12 μg/mL, respectively. The method was successfully applied to the simultaneous determination of 16 analytes in 15 samples of P. notoginseng collected from different places of China, which indicated that multiple columns HPLC can be used for comprehensive quality control of P. notoginseng.