Putative Nicotiana glauca (wild tobacco) poisoning was diagnosed in a flock of ostriches near Oudtshoorn, South Africa. Post mortem examinations (n = 7) were performed on ostriches (Struthio camelus) that had died. Suspicious leaf remnants (weighing 80–770 g), packed in a layer on top of other plant material, were carefully separated from the proventricular content and submitted for chemical determination of anabasine, the major toxic principle contained by this plant. A standard solid phase extraction method was used followed by an optimised liquid chromatography-mass spectrometry procedure. Anabasine was detected in the leaf remnants (114–177 μg/g dry weight) removed from the proventriculus of the ostriches that succumbed as well as in control N. glauca leaves (193 μg/g dry weight). The analytical methods used in this study revealed the presence of anabasine in the suspicious leaf remnants, indicating that the birds had been exposed to N. glauca and had died of this poisoning.