The accidental ingestion of leaves or cut branches of oleander (Nerium oleander) often results in severe poisoning in cattle. This natural shrub contains oleandrin, a highly toxic cardiac glycoside, which may prove fatal even at very low amounts. For this reason, a highly sensitive ultra-high performance liquid chromatography-tandem mass spectrometry method was developed and validated for in vivo oleandrin determination in bovine blood and serum samples or in bovine liver and heart for post-mortem diagnosis. The method was highly selective as the mass spectrometer worked in selected reaction monitoring mode using the first five most intense m/z transitions of oleandrin. The limits of detection and quantitation were 0.11 and 0.36 ng mL⁻¹, respectively, whereas the method detection and quantitation limit values were in the range of 0.006–0.051 ng g⁻¹ and 0.018–0.154 ng g⁻¹, respectively. The chromatographic run was only 5 min and the time employed for the sample pretreatment (25–30 min) is useful to avoid the presence of matrix effects. All the recovery values were within 62.9–80.5%. The method was successfully used to analyse incurred samples from an accidentally poisoned cow and is expected to be applicable to forensic purposes.