The aim of the research was to develop a new sensitive method for simultaneously the determination of psychoactive drugs: 1-benzylpiperazine, 7-aminoclonazepam, alprazolam, clonazepam, diazepam, estazolam, lorazepam and tetrazepam in human serum and hair samples. In the preparation step, microwave-assisted extraction (MAE) was used. Extracts were analyzed by means of capillary electrophoresis with mass spectrometry time-of-flight detection (CE-TOF-MS). In the validation study of the MAE/CE-TOF-MS analytical method, three concentration levels of analytes (10, 100 and 250 ng/mL for serum and 0.2, 2.2 and 5.6 ng/mL for hair) were taken into account. Such parameters as limit of detection (0.4-1.2 ng/mL for serum, 6.0-23.0 pg/mg for hair), limit of quantification (1.3-4.1 ng/mL for serum, 20.0-77.0 pg/mg for hair), precision (3.0-11.3% for serum, 2.4-14.2% for hair), accuracy of the assay (RE) (-8.0 to 12.0% for serum, -8.0 to 11.0% for hair), recovery (88.6-113.4% for serum, 86.1-107.4% for hair) and matrix effects (87.9-110.7% for serum, 85.1-108.4% for hair) were calculated for the studied compounds. Then, the MAE/CE-TOF-MS method was successfully applied to the analysis of hair samples taken from patients treated with benzodiazepines.