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ETHNOPHARMACOLOGICAL RELEVANCE Paris polyphylla var yunnanensis as a traditional Chinese medicine has been used in the treatment of liver disease for thousands of years. Rhizoma Paridis saponins (RPS) were the main active ingredients in Paris Polyphylla with an excellent antitumor effect. However, metabolic and distribution of RPS has not been known. AIM OF THE STUDY The objective of this study was to research metabolic and distribution of RPS. MATERIALS AND METHODS In this study, the separation and simultaneous determination of RPS in rat plasma and tissues were developed and validated by LC-MS/MS. The permeability and recovery of RPS were tested by Caco-2. S9 assay suggested the metabolic mode of RPS in rats. RESULTS After oral administration of RPS, the metabolic compound like diosgenin was detected in different tissues although there was none in RPS. The concentration of PI, PII, PVI, PVII, PH and gracillin in the spleen was the highest among these organs. The content of diosgenin were the highest in lung and brain. Caco-2 test indicated that PI, PII, PVI and PVII were low permeability and low recovery. Efflux ratio indicated that PVI should be a potential P-gp substrate. Potential P-gp substrate may be PVI. S9 assay suggested that RPS possess slow metabolic and moderate metabolic compounds. CONCLUSIONS Integrated LC-MS/MS analysis of serum samples, together with Caco-2 and S9 assays provided a theoretical basis for the application of RPS in the future.