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Cerebrovascular reactivity (CVR), which denotes brain’s vasodilatory capacity, is broadly utilized in cerebrovascular and neurodegenerative diseases. However, the most popular hypercapnia method in human studies is unsuitable for small animals due to the difficulty in measuring end-tidal CO2. Here, we took a different approach using a pharmacological vasodilatory stimulus, acetazolamide. Plasma level of acetazolamide and vascular responses were quantified by high-performance liquid chromatography and perfusion MRI, respectively. Evidences of feasibility, safety, temporal characteristics, and dose-dependence have been demonstrated. This new CVR technique may open several avenues for preclinical research on cerebrovascular diseases and therapeutic testing in different animal models.