A fast high-performance liquid chromatography (HPLC) coupled with electrospray ionization (ESI) tandem mass spectrometry method was developed to determine 24 components including 11 phenolic compounds, 9 phenolic amides, and 4 cyclic peptides in Cortex Lycii. The analytes were quantified by a triple quadrupole instrument in multiple reaction monitoring (MRM) mode. The fragmentation patterns of phenolic amides and cyclic peptides using ESI and collision-induced dissociation (CID) techniques are reported. This assay method was validated with respect to linearity (r2>0.9920), precision, repeatability, and accuracy (recovery rate between 93.0 and 105.9% with RSD<4.4%). The analytical results of 28 batches of Cortex Lycii indicated that cyclic peptides and phenolic amides were not only the abundant constituents, but also the characteristic components for Cortex Lycii to distinguish from the adulterants. Principle component analysis (PCA) was used to discriminate samples from different geographical regions of China, and cyclic peptides were considered to be the chemical markers responsible for the classification. The systematic and integrated assessment of Cortex Lycii provides sufficient evidence for the establishment of the quality standard.