The liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (LC-ACPI-MS) method of glucocorticosteroids determination proposed in this paper should be considered as the simplest and at the same time an adequately sensitive and specific method for the purpose of posthumous diagnostics, especially when biochemical confirmation of intravital stress agent activity is important in an investigated case. In this study, a LC-MS assay was developed and optimized for the determination of cortisol, cortisone, and corticosterone. In order to separate analytes from the biological matrix, the liquid-liquid extraction method was applied. Post-mortem blood was extracted with dichloromethane in alkaline medium (pH 6.5). The MS was operated in ACPI mode, and quantitative analysis was carried out in the selected ion monitoring mode (SIM). Quantitative analysis was carried out on the basis of analytical signals obtained from positive pseudomolecular ions of the determined substances (cortisol, cortisone, and corticosterone). Method validation was performed. Figures, tables, and references