A high sensitive performance liquid chromatography-electrospray ion trap-mass spectrometric（LC-MSn） method was developed for the determination of the major methyl protodioscin metabolites（MPD） in rat’s plasma，urine，bile and feces in order to discover its in vivo metabolism pathway.The Sprague-Dawley rats were given a single intravenous injection of 40 mg/kg MPD，and 30 min blood sample，24 hour bile sample，48 hour urine sample and feces samples after administration were collected.The in vivo samples were extracted by solid phase extraction（SPE）.The extracted samples were separated with Phenomenex Gemini C18 column，using methanol（B）-water（A） as mobile phase by elution gradient.The flow rate was set at 1.0 mL/min.The column temperature was 30 ℃.Total of fourteen metabolites in rat biosamples were detected under negative and positive modes .Six of them were identified by comparing HPLC retention times and mass spectra with those of authentic standards.They were protodioscin（M3），26-O-β-D-glucopyrannosyl-（25R）-furan-5-ene-3β,22α,26-trihydroxy-3-O-α-L-rhamnopyranosyl-（1→2 or 4）-β-D-glucopyranoside（M5），MPD（M0），Pseudoprotodioscin（M7），26-O-β-D-glucopyrannosyl（25R）-furan-5-ene-3β,26-dihydroxy-22-methoxy-3-O-α-L-rhamnopyranosyl-（1→ 4）-β-D-glucopyranoside（M9） ，26-O-β-D-glucopyrannosyl（25R）-furan-5-ene-3β,26-dihydroxy-22-methoxy-3-O-α-L-rhamnopyranosyl-（1→2）-β-D-glucopyranoside（M10） and dioscin（M11）.Five of them were proposed based on the MS fragmentation characteristics of metabolite contrast.The results indicated that protodioscin and dioscin were two primary metabolites，and the formation routes of these two metabolites were the major metabolic pathways of MPD in rat.Deglycosylation was found to be the primary reaction.Most of the metabolites belong to phase I,except 2-metabolites belong to phaseⅡreaction and most of the structure changes took place in sugar part rather than in mother nucleus.