In this study, HPLC-ESI-MS and HPLC methods were established to explore the differences in the main chemical components and content of Mori Cortex with(mulberry root bark) and without(Mori Cortex) the phellem layer from both qualitative and quantitative aspects. The HPLC-ESI-MS method was used for quality analysis in positive and negative ion modes, and 33 compounds were identified in mulberry root bark, 22 compounds in Mori Cortex, and 26 compounds in phellem layer; mulberry root bark and Mori Cortex shared 22 components, and mulberry root bark has 11 unique compounds; Mori Cortex and its phellem layer shared 15 components, while Mori Cortex has 7 unique compounds. HPLC method was used to simultaneously determine 7 major constituents, including mulberroside A, chlorogenic acid, dihydromorin, oxyresveratrol, moracin O, kuwanon G, and kuwanon H, and the developed method showed good linearity(r>0.998 9) within the concentration range and the recoveries varied from 99.88% to 103.0%, and the RSD was 1.7%-2.9%. The HPLC results showed that the contents of the 7 compounds have great differences in 13 batches samples, compared with mulberry root bark, the contents of mulberroside A, chlorogenic acid, dihydromorin and moracin O of Mori Cortex were increased, while the contents of oxyresveratrol, kuwanon G and kuwanon H were decreased after peeling process. These results can provide a basis for the rationality and quality control of Mori Cortex required to remove the phellem layer.