The number of scratching bouts is generally used as a standard method in animal models of itch. The aim of the present study was to validate the application of infrared thermography (IR-Th) in a serotonin-induced itch model in rats. Adult Sprague-Dawley male rats (n = 24) were used in 3 consecutive experiments. The first experiment evaluated vasomotor response (IR-Th) and scratching behavior (number of bouts) induced by intradermal serotonin (10 μl, 2%). Isotonic saline (control: 10 μl, 0.9%) and Methysergide (antagonist: 10 μl, 0.047 mg/ml) were used. The second experiment evaluated the dose-response effect of intradermal serotonin (1%, 2% and 4%) on local temperature. The third experiment evaluated the anesthetized rats to test the local vasomotor responses in absent of scratching. Serotonin elicited significant scratching and lowered the local temperature at the site of injection. A dose-response relationship of serotonin and local temperature changes was found by IR-Th. There was a significant negative correlation between temperature and number of scratching bouts. Vasoregulation at the site of serotonin injection occurred in the absent of scratching reflexes. In conclusion, IR-Th is a reliable, non-invasive, and objective method for assessment of serotonin-induced itch model in rat. This method may prove benefits in studying pruritus and antipruritic agents.