Because of the recognized inaccuracy and unreliability of currently available methods for the quantification of histamine in biological fluids, a method for quantification of urinary histamine by stable isotope dilution assay with negative ion chemical ionization mass spectrometry has been developed. Following the addition of [ 2H 4]histamine to 1 ml of urine, histamine is extracted into butanol, back-extracted into HCl, derivatized to the pentafluorobenzyl derivative (CH 2C 6F 5) 3-histamine, extracted into methylene chloride, and then quantified with negative ion chemical ionization mass spectrometry by selected ion monitoring of the ratio of ions m z 430 434 . Twenty samples can be assayed in 2 days. Precision of the assay is ±2.7% and the accuracy is 97.6%. Lower limits of sensitivity are approximately 100–500 fg injected on-column. This assay provides a very sensitive, accurate, and efficient method for the quantification of histamine in human urine.