Objective To use high-performance liquid chromatography-time of flight mass spectrometry (HPLC-TOF/MS) for examining the steroidal saponins in rat urine after oral administration of Paris polyphylla extract, so as to lay a foundation for studying the metabolism of steroidal saponins in vivo. Methods SD rats were administered with an oral dose of 1.6 g Paris polyphylla extracts/kg body weight. The urine samples were collected 24 h after administration by oral gavage. The sample analysis was carried out on a reverse phase MG-C₁₈ column (3.0 mm×100 mm, 3.0 μm) using a gradient mobile phase system of acetonitrile-water containing 0.1% formic acid. TOF/MS was applied for qualitative analysis under positive and negative ion modes. The steroidal saponins in rat urine were identified by using the accurate molecular weight obtained by TOF/MS and formula database. Results A total of 20 steroidal saponins were identified in the rat urine, and two pairs of isomers were deduced through their fragment ions and standards: polyphyllin Ⅶ and pennogenin-3-O-α-L-rhamnopyranosyl(1→4)-α-L-rhamnopyranosyl (1→3)-\[α-L-rhamnopyranosyl(1→2)\]-β-D-glucopyranoside; and gracillin and pennogenin-3-O-α-L- rhamnopyranosyl(1→4)-α-L-rhamnopyranosyl(1→4)-β-D-glucopyranoside.Conclusion The established analysis method is accurate, reliable for identifying steroidal saponins in vivo, which paves a way for further pharmacokinetics and pharmacodynamics study of Paris polyphylla.