[Subject]A test substance application system for animal experiment capable of administering a required amount of a test substance uniformly and reliably from a nose or a mouth into a nasal cavity or into a lung of an experimental animal is provided.[Means for Solution]The system includes a respiration monitoring device 2 that monitors a respiration state of an experimental animal detected by a respiration pick-up device 1 to thereby measure a timing upon switching from an expiratory phase to an inhalatory phase and outputs a trigger signal T at that timing, and an application device 3 that sprays a predetermined amount of a test substance into a nasal cavity or an oral cavity of the experimental animal when the trigger signal T is outputted from the device 2.