Abstract: In this paper, water extract of Nauclea officinalis was absorbed by AB-8 macroporous resin and subsequently gradient-eluted with alcoholic solution of different proportion to prepare pumiloside monomer of high purity. The metabolites in urine, feces and bile of rats with gavage administration were analyzed by UPLC-QTOF/MS. Accurate MS/MS data of all components were collected with full scan mode, and analyzed by MetaboLynx software. Results showed that the monomer of high-purity pumiloside was prepared and four metabolites in rats were identified.