A sensitive and selective method has been developed for simultaneous determination of triazolam and its major metabolites, alpha-hydroxytriazolam and 4-hydroxytriazolam, in human whole blood and urine. The drugs were effectively extracted using a 3-step solvent extraction procedure followed by tert- butyldimethylsilyl derivatization, and subjected to gas chromatography-mass spectrometry in the negative ion chemical ionization mode. Estazolam was used as an internal standard. The calibration curve for each compound was linear in the range from 0.5 to 100 ng/g, and the lower limit of detection was 0.1 ng/g for whole blood and 0.2 ng/g for urine. Within-day precision of this method was evaluated in whole blood and urine samples at the concentration of 10 ng/g; the coefficient of within-day variation ranged from 2.7 to 10.8%.