Described in this unit is the stress-induced hyperthermia (SIH) test in mice in a singlehoused format. This protocol has proven reliable in detecting the anxiolytic properties of test compounds. In this test, SIH is quantified in singly housed mice using a rectal temperature measurement as the stressor. Rectal temperature is measured twice at a 10-min interval. Due to the stress experienced during the first temperature measurement, the temperature of the second measurement (T 2 ) is ∼0.8° to 1.5°C higher than that of the first (T 1 ). This difference in temperature (ΔT = T 2 - T 1 ) is defined as the SIH response. The SIH response is reduced by different classes of anxiolytics. The SIH test is simple and robust, it does not require training of animals, and test compound effects on motor behavior, feeding, and nociception do not affect test outcome. Furthermore, it is one of few anxiety tests that focuses on the physiological component of anxiety. © 2009 by John Wiley & Sons, Inc.