Some plant species are known to cause calcium intoxification in grazing animals. This has been attributed to the presence of vitamin D3-like activity. However, research into the presence of vitamin D3 in plants has been limited. One reason for this may be limitations in the analytical methods available for unambiguous detection and quantification of vitamin D3. This paper presents a new method for determining vitamin D3 and its sterol precursors. The method is based on saponification and extraction followed by solid phase clean-up of the compounds from plant leaves and detection by APCI-MS. Recoveries ranged from 101% to 114% and precision from 3% to 12%. Detection limits were 2–8 ng/g fresh weight for the substances tested. In a pilot study we found that Solanum glaucohyllum Desf. and Solanum lycopersicum L. produced vitamin D3 after UV-treatment. The preliminary results presented suggest that vitamin D3 formation in plants is dependent on light exposure.