Objective An analytical approach has been studied lor high performance liquid chro-matographic determination of Diphacinone and its metabolites extracted from urine. Method Diphacinone and its metabolites extracted from rabbit urine by solid phase extraction (SPE) cartridges (CN and SAX) were analyzed with coumarin as the internal standard. Diphacinone and its metabolites were separated by reversed-phase gradient chromatography with DAD detector at 311nm. The analytical column was Hypersil BDS C18 (150mm?4.6nmi. d. ) and the guard column was Phenomenex ODS (4mm?3.0mmi. d.). The mobile phase was a gradient mixture of aqueous solution (A) and methanol solution (B), both containing 0.5% ion pair A. Results Eleven diphacinone metabolites were detected in poisoned rabbit urine by this method. Conclusion This method is simple, exact and rapid.