Abstract: HPLC-MS/MS was applied to the determination of oleandrin and adynerin in blood. The blood sample was dispersed and affixed on solid phase supported liquid-liquid extraction column which was then eluted with ethyl acetate. The eluate was then separated by using Kinetex C₁₈ chromatographic column as stationary phase, and mixtures of 10 mmol·L^-1 ammonium formate solutioncontaining 0.1% (φ) formic acid and acetonitrile in various ratios as mobile phase in gradient elution. ESI⁺ and multi-reactions monitoring were adopted in MS/MS. Linear relationships between values of peak area and mass concentration of oleandrin and adynerin were kept in the same range of 2-100 μg·L^-1, with same detection limit (3S/N) of 0.5 μg·L^-1. On the base of blank sample, test for recovery was made by standard addition method; values of recovery found were in the range of 90.0%-98.0%, with RSD's (n=6) in the range of 2.1%-7.3%.