Simultaneous determination of oleandrin and its three related compounds, desacetyloleandrin, oleandrigenin, and gitoxigenin in blood by using liquid chromatography-three-dimensional quadrupole mass spectrometry (LC-3DQMS) system equipped with sonic spray ionization (SSI) interface was conducted. This analyzing method was suitable for all of these compounds except gitoxigenin. The limits of detection of oleandrigenin and desacetyloleandrin from blood were 2 ng/mL and that of oleandrin was 3 ng/mL. The calibration curves for oleandrin, desacetyloleandrin, and oleandrigenin were linear in the range of 5-100 ng/mL. The coefficients of variation of oleandrin, desacetyloleandrin, and oleandrigenin in the blood were satisfactory ranging from 1.6% to 4.1%. This analysis method was applied to a fatal case of oleander poisoning. As a result of liquid chromatography-mass spectrometry (LC-MS) analysis, oleandrin was detected in heart blood and cerebrospinal fluid. Desacetyloleandrin, oleandrigenin, and gitoxigenin were not detected. In order to make identification of oleandrin reliable, LC-MS-MS analysis was performed. The concentrations of oleandrin found in the heart blood and cerebrospinal fluid were 9.8 and 10.1 ng/mL, respectively.