An analytical method was developed and validated for the measurement of the novel analgesic and anti-inflammatory drug robenacoxib in blood and plasma of dogs and cats. To prevent nonreproducible carry-over effects, an initial solid phase extraction procedure was followed by high pressure liquid chromatography analysis for samples with concentrations in the range 500 to 20 000 ng/mL. To improve accuracy, samples of concentration 3 to 100 ng/mL were analyzed by liquid chromatography-mass spectrometry. Applying these methods, blood concentration-time profiles and pharmacokinetic variables of robenacoxib in dogs were determined in a four-phase cross-over study, which compared different routes of administration of the drug, including intravenous (i.v.) injection, oral application with and without feed, and subcutaneous (s.c.) application. After i.v. administration the mean clearance from blood was 0.81 L/kg/h, the volume of distribution was 0.77 L/kg for the elimination phase and 0.24 L/kg for steady-state, and the terminal half-life in blood was 0.63 h. Maximum blood concentrations were obtained in less than 1 h following oral or s.c. application. Absolute bioavailability was 88% after s.c. injection, 84% after oral administration to fasted dogs, but was reduced to 62% when applied orally to fed dogs. In canine and feline plasma the degree of binding of robenacoxib to plasma protein in vitro was greater than 98%. The blood:plasma concentration ratio was 0.44:1 in the dog and 0.65:1 in the cat. In conclusion analytical methods for the quantification of robenacoxib in blood and plasma in the dog and cat were developed and validated. In dogs, robenacoxib has good bioavailability after oral (84%) and subcutaneous (88%) administration.