Perphenazine enanthate has been used in wild animals as a tranquilizer during the period of adaptation to new environments to reduce stress, mortalities and injuries. A gas chromatographic procedure for the quantitative measurement of perphenazine in otter urine has been developed and validated. The method involved an enzymatic hydrolysis with beta-glucuronidase-arylsulfatase from Helix pomatia, followed by a solid-phase extraction with Bond Elut Certify cartridges. The resulting organic phase was evaporated, and the dry extract was derivatised with MSTFA to form the O-TMS derivative. The derivatised extracts were analysed by gas chromatography-mass spectrometry using SIM acquisition mode, measuring three diagnostic ions (m/z 246, 372 and 475). Another phenothiazine derivative, fluphenazine, was used as the internal standard (I.S.). Extraction recoveries for perphenazine and I.S. were 87.6 +/- 8.2% (n=4) and 106.7 +/- 13.4% (n=4), respectively. The calibration curves were linear in the range from 4 to 100 ng/ml (r2=0.99). The limits of detection and quantification were estimated as 1.2 and 3.5 ng/ml, respectively. Precision and accuracy obtained in intra-assay studies were in the ranges of 1.3-8.7 and 1.7-19.5%, respectively, using control samples containing 6, 16 and 60 ng/ml of perphenazine. In inter-assay experiments, precision ranged from 4.3 to 14.9% and accuracy from 3.1 to 11.8%. Examples of the application of the perphenazine quantification method in otter urines after administration of perphenazine enanthate are presented.