Brain death diagnosis may be confounded by centrally acting drugs. The certainty of brain death diagnosis can be enhanced by demonstrating that the concentrations of such drugs are well below the therapeutic range. A combined high-performance liquid chromatography-based method was developed for the benzodiazepines midazolam, 1-hydroxymidazolam, 1-hydroxymidazolam glucuronide, diazepam, and nordiazepam and for the barbiturates thiopentone and pentobarbitone in serum or plasma of critically ill patients. The lower limits of detection of the assays for benzodiazepines and barbiturates were 2.5 ng/mL and 0.05 microg/mL. The lower limits of the working ranges of these assays were set at 25 ng/mL and 0.5 microg/mL, respectively, and are below the lowest pharmacologically active plasma concentrations of these drugs. Intra- and interday coefficients of variations were less than 2.5% and 11.0% throughout, as determined with six replicates (n = 6). These assays were accurate in that the relative difference between actually measured and expected concentration never exceeded 12%. Utilization of these assays will render the diagnosis of brain death more reliable.