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Introduction: Polygoni Multiflori Radix (PMR) is commonly used in traditional medicine as both raw and processed forms. Raw PMR was prepared into processed PMR via processing procedure; however, there is a lack of standardized protocols ensuring the completeness of processing., Objective: This aims to develop a strategy based on a metabolomics approach for the comprehensive chemical profiling and comparison of raw and processed PMR and establish a basis for PMR processing standardization., Materials and Methods: Methanol extracts of raw and processed PMR were fractionated by centrifugal partition chromatography (CPC) with an optimized two-phase solvent system based on the partition coefficient calculated from the shake-flask method to produce primary (1°Ms)- and secondary metabolites (2°Ms)-enriched fractions. These fractions were profiled by 1D and 2D and selective 1D NMR experiments, spectral fitting, and comparison with reference standards. The profiled compounds were quantified via quantitative 1 H NMR (qHNMR) to show the chemical changes, which were correlated with changes in antioxidant effects on H2452 cells., Results: A CPC method was developed to efficiently separate 1°Ms- and 2°Ms-enriched fractions. This method achieved high purity of the major stilbene in PMR in a single run. qHNMR effectively quantified four 2°Ms and twenty-one 1°Ms in both raw and processed PMR, including meso-butane-2,3-diol, which was first reported from processed PMR. Changes in chemical composition of PMR because of processing are highly correlated to the increase of antioxidant activity., Conclusion: A convenient and cost-effective strategy for the comprehensive chemical profiling of raw and processed PMR was developed by combining countercurrent separation and qHNMR. This approach will contribute to the standardization of medicinal herbal materials., (© 2024 John Wiley & Sons Ltd.)