Abstract: The petals of Hemerocallis middendorfii Trautv. et Mey. and H. ‘Pardon Me’ were used to study the sample preparation method of carotenoids, and the samples were qualitatively and quantitatively analyzed by UPCC-MS. Results showed that:(1)In the process of carotenoids sample preparation, different extraction solvents, shaking methods and saponification methods have significant influence on the extraction efficiency of carotenoids, and by multi-factor analysis of variance, the optimal extraction method was determined as follows:B acetone:hexane(3:5/V:V) as extraction solvents, extracted in a shaking incubator and saponification 16 h at room temperature; (2)UPCC-MS technology can separate the carotenoids in samples efficiently in 10 min, and with lesser use of toxic chemicals, UPCC-MS can considered to be a good choice for analyzing carotenoids; (3)There are 20 kinds of carotenoids in H.middendorfii and H. ‘Pardon Me’ petals, and these two kinds of materials with different colors had different composition and content of carotenoids.