Tramadol is a centrally acting analgesic drug used in veterinary and human clinical practice. Its metabolism has been largely characterized in human being but is still long to be comprehended in several animal species, especially in the dog. The aim of the present study was to develop and validate a new analytical procedure to investigate HPLC the metabolization/elimination process tramadol in urine of dogs by HPLC-FL or HPLC-MS/MS. A single oral dose of tramadol (4mg/kg) was administered to 4 male Beagle dogs and the urine was naturally collected. This matrix either hydrolyzed than un-hydrolyzed was extracted with different blends of solvents to detect the total or free form of the analytes, respectively. The present method allowed to obtain good selectivity, accuracy, precision and recoveries without the need of time consuming or expensive clean up steps. The short chromatographic time courses allowed this analysis to be proposed for routine purposes. The HPLC-MS/MS detected in the urine two metabolites (M6 and M7) considered negligible in humans. The low LOQ showed that the method could be useful for the determination of the illegal use of this drug in race-dogs' urine.