This study evaluated the Abbott ADx and TDx serum benzodiazepine assays for the analysis of alprazolam in serum. These systems were compared to the EMIT tox serum benzodiazepine assay and a gas chromatographic (GC) method. Six-point calibration curves (nordiazepam concentrations from 50-1,000 ng/mL) were stable on both instruments for at least 14 days. The precision of these assays was determined using three control serums containing 75,300, and 700 ng/mL of nordiazepam. On the ADx, within-run coefficients of variation (CV) for the three controls were 3.9, 3.1, and 2.8%, respectively. Between-run CVs were 2.8, 1.4, and 1.2% on the ADx. For the TDx, within-run CVs were 1.9, 1.4, and 2.6, and between-run CVs were 4.1, 1.3, and 1.9% for the three controls. Percent cross-reactivity of serum alprazolam calibrators (25-100 ng/mL) ranged from 77-88% on the ADx and 60-79% on the TDx. 99 serum specimens containing alprazolam were analyzed with the ADx, TDx, EMIT, and by gas chromatography-electron capture detection (sensitivity of 5 ng/mL). Overall, 48 of 99 specimens on the TDx, 43 of 99 on the ADx (both with a cutoff of 12 ng/mL), and 0 of 99 by EMIT (cutoff of 300 ng/mL) were positive. Of the specimens reading negative on the Abbott systems, 43 of 51 TDx results and 44 of 56 ADx results were obtained in specimens containing less than 25 ng/mL of alprazolam. For specimens with more than 25 ng/mL of alprazolam in serum (by GC), 8 specimens were negative on the TDx and 12 on the ADx.