As an approved medicinal product and reportedly an abused substance that have been associated with death and "considered to be a factor...of impaired driving, sexual assault, and other violent crimes", triazolam is controlled at the same level (Level III) as flunitrazepam in Taiwan. Alleged misuses of this substance have been associated with case specimens submitted to this laboratory. A sample preparation (with and without enzymatic hydrolysis) and gas chromatography-mass spectrometry protocols were evaluated and applied to the analysis of free and total alpha-hydroxytriazolam (the main metabolite of triazolam) in urine. Ions designated for TMS-derivatized alpha-hydroxytriazolam and alpha-hydroxytriazolam-d4 are m/z 415, 417, and 430 and 419, 421, and 434, respectively. The overall protocol achieved the following results when applied to the analysis of 2-mL drug-free urine specimens fortified with 10-200 ng/mL alpha-hydroxytriazolam: recovery, 95%; interday and intraday precision ranges, 1.50-3.52% and 0.93-4.71%, respectively; linearity, r2 > 0.99; and limits of detection and quantitation, 0.05 and 0.1 ng/mL, respectively. This protocol was applied to the analysis of case specimens and urine samples collected from two patients (A and B) taking one oral dose of Halcion (0.25 mg triazolam). Excretion profiles of free and total alpha-hydroxytriazolam show that free alpha-hydroxytriazolam is detectable, but at very low levels (< 5 ng/mL). Peak excretion of total alpha-hydroxytriazolam occurs at approximately 5-10 h following the drug intake. Total alpha-hydroxytriazolam is excreted at detectable levels approximately 2-35 h following an oral dose of 0.25 mg triazolam. Total free and conjugated alpha-hydroxytriazolam excreted by A and B are 0.61% and 31.6%; and 0.36% and 57.2% of the dose, respectively.