Tetrahydrobiopterin in the murine locus coeruleus was measured as its fully oxidized form, biopterin, using a HPLC coupled to a fluorescence detector, because tetrahydrobiopterin itself cannot be detected by such means. The differential oxidization method distinguished tetrahydrobiopterin-derived biopterin and dihydrobiopterin-derived biopterin. The protocol reported here is a rapid and sensitive method that facilitates the measurement of tissue and/or cellular tetrahydrobiopterin. Using this assay protocol, we were able to detect and quantify variations in the tetrahydrobiopterin content in the murine locus coeruleus.