Prostaglandin D₂ (PGD₂) has been found to be an important pathophysiological mediator in a number of human disorders. Thus a means to assess the endogenous production of PGD₂ is of considerable clinical value. To accomplish this goal, we developed a method for the quantification of the major urinary metabolite of PGD₂, 9α, 11β-dihydroxy-15-oxo-2,3,18,19-tetranorprost-5-ene-1,20-dioic acid, by gas chromatography/negative ion chemical ionization mass spectrometry. This metabolite was chemically synthesized and converted to an ¹⁸O₄-labeled derivative for use as an internal standard. Novel derivatization and purification procedures were incorporated in the assay taking advantage of the ability of the lower side chain of this molecule to undergo cyclization at acidic pH to form a hemiketal, γ-lactone, and uncyclization with methoximation. Precision of the assay is ±7% and accuracy is 96%. The lower limit of sensitivity is approximately 50 pg. Normal levels for the urinary excretion of this metabolite in 18 normal adults was found to be 1.08 ± 0.72 ng/mg creatinine (mean ± 2SD). Substantial elevations in the urinary excretion of the metabolite were found in clinical situations in which prostaglandin D₂ has been shown to be released in increased quantities. Thus, this assay provides a sensitive and accurate method to assess endogenous production of prostaglandin D₂ as a means to explore the pathophysiological role of prostaglandin D₂ in human disease.