Withanolides from six parts (flower, leaf, stem, root, seed, and peel) of Datura metel L. (D metel L.) obtained from ten production areas in China were identified and quantified by UPLC-MS/MS. A total of 85 withanolides were characterized for the first time using the UPLC-Q-TOF-MS/MS system. Additionally, a simultaneous, rapid and accurate measurement method was developed for the determination of 22 bioactive withanolides from ten production areas with the UPLC-Q-TRAP-MS/MS system. The results show the total withanolide content is highest in the leaves (155640.0 ng/g) and lowest in the roots (14839.8 ng/g). Compared with other production areas, the total content of plants from Dujiangyan was the highest at 82013.9 ng/g (value range of ten areas: 82013.9–42278.5 ng/g). The results also show significant differences in the distribution of withanolides in the different plant parts, as well as across different production areas. This is a breakthrough report providing a simultaneous qualitative and quantitative analysis of 22 withanolides in D. metel L. It could be the basis for the more rational use of various parts of D. metel L., and the expansion of medicinal resources. This work also lays a solid foundation for research on the quality control of D. metel L.
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