Fluorophotometric method for the determination of dehydroepiandrosterone sulfate in urine has been established. The method involves the formation of an ion-pair complex between the sulfate and acridine orange, and extraction of the complex into 1, 2-dichloroethane, followed by the measurement of the fluorescence intensity at λ_ex 495 nm and λ_em 520 nm. The limit of detection was about 80 ng/ml of dehydroepiandrosterone sulfate and the recovery from urine was between 92.3 and 103.3%. The present method has been applied to the determination of dehydroepiandrosterone sulfate present in normal human urine.