Dogs undergo various surgical procedures such as castration, ovariohysterectomy, and other orthopedic procedures, which are known to cause inflammation and pain. Non-steroidal anti-inflammatory drugs (NSAIDs) are very effective analgesics for alleviating postoperative pain in veterinary medicine. Ketoprofen is currently approved in Australia and the United States for treating different painful conditions in dogs. This study evaluated the pharmacokinetic parameters of ketoprofen after intravenous (IV) and transdermal (TD) administration in healthy dogs. A novel transdermal ketoprofen (TDK) formulation containing 20% ketoprofen, dissolved in a combination of 45:45% isopropanol and Transcutol, along with 10% eucalyptus oil, was developed and evaluated for in vitro dermal permeation using Franz diffusion cells. A crossover study was then conducted to determine the pharmacokinetic parameters of the formulation in six dogs following IV ketoprofen (1 mg/kg) and TDK (10 mg/kg) administration. A liquid chromatography-mass spectrometry (LC-M/MS) method was used to measure plasma concentrations of ketoprofen over time, and a non-compartmental analysis determined the pharmacokinetic parameters. The mean terminal elimination half-life ( h), (µg·h/mL), and mean residence time (MRT, h) between IV and TDK groups were 4.69 ± 1.33 and 25.77 ± 22.15 h, 15.75 ± 7.72 and 8.13 ± 4.28 µg·h/mL, and 4.86 ± 1.81 and 41.63 ± 32.33 h, respectively. The calculated bioavailability (%) was ~7%, with a lag time of 30 min to achieve effective plasma concentrations after the application of TDK.