Alprazolam is a benzodiazepene, which is used for treatment of anxiety and other disorders. Two metabolites of alprazolam, 4-OH-alprazolam and α-OH-alprazolam, have been shown to be active. To accurately study the pharmacokinetics, all three analytes should be quantified from plasma. Under new dose regimens with alprazolam, concentrations of 4-OH-alprazolam and α-OH-alprazolam could b e <0.05 ng/mL in plasma 24 hours post-dose. Previous LC/MS/MS assays quantified alprazolam and only α-OH-alprazolam to the 0.05 ng/mL level. Other assays quantified the drug and both metabolites to the 0.05 ng/mL level; however, the assay suffered from poor precision (>15% RSD). Here we report on an accurate and precise validated LC/MS/MS assay for the determination of alprazolam, α-OH-alprazolam and 4-OH-alprazolam from human plasma.