Er-Zhi-Pill, which consists of Ligustri lucidi fructus and Ecliptae prostratae herba, is a classical traditional Chinese medicinal formulation widely used as a liver-nourishing and kidney-enriching tonic.To identify the bioactive ingredients of Er-Zhi-Pill and characterize the variation of chemical constituents between co-decoction and mix of individually decocted L.lucidi fructus and E.prostratae herba, a novel metabolomics approach based on ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry in both positive and negative ion modes, was established to comprehensively analyze chemical constituents and probe distinguishable chemical markers.In total, 68 constituents were unambiguously or tentatively identified through alignment of accurate molecular weights within an error margin of 5 ppm, elemental composition and fragmentation characteristics, including 8 constituents which were confirmed by comparing to reference standards.Furthermore,principal component analysis and partial least squares discriminant analysis using Simca-p+ 12.0 software were applied to investigate chemical differences between formulations obtained by co-decoction and mix of individual decoctions.Global chemical differences were found in samples of two different decoction methods, and 16 components, including salidroside, specneuzhenide and wedelolactone, contributed most to the observed differences.This study provides a basic chemical profile for the quality control and further mechanism research of Er-Zhi-Pill.