In addition to total anamnesis, one of the important aspects in diagnosis of brain death is the exclusion of effective plasma concentrations of drugs that might mimic brain death. A minimum consensus for toxicological analysis in this context includes relevant analytes (thiopental, pentobarbital, methohexital, phenobarbital, diazepam, nordazepam, and midazolam) and proposes limits of quantification. Propofol is another relevant drug. After liquid-liquid extraction of 200 microL of plasma using 50 microL of a solution of deuterated internal standards in butyl acetate and 50 microL of butyl acetate, 2 microL of the organic phase was analyzed by gas chromatography-mass spectrometry using selected-ion monitoring mode. Validation included the parameters selectivity, calibration model, precision and accuracy, and extraction efficiency. Accuracy and precision data obtained using 6-point and 1-point calibration were compared. The abovementioned analytes were separated within 10 minutes and sensitively detected. No interfering peaks were observed in blank samples from 10 different sources. The linearity ranges were 0.5-6 mg/L for propofol, 0.25-10 mg/L for pentobarbital and thiopental, 0.125-10 mg/L for methohexital, 2.5-50 mg/L for phenobarbital, 0.05-2.5 mg/L for diazepam and nordazepam, and 0.01-0.5 mg/L for midazolam. Extraction efficiency ranged from 85% to 111%. The acceptance criterion for accuracy and precision (99% confidence interval of measured mean within +/-50% of target value) was fulfilled for all analytes, even with 1-point calibration using a calibrator close to the center of the linearity range. The assay was applied to analysis of real brain death cases. In conclusion, the described assay allowed fast and reliable determination of analytes relevant to diagnosis of brain death, and 1-point calibration kept the workload low.