Background: Raw Moutan Cortex (RMC), derived from the root bark of Paeonia suffruticosa, and Processed Moutan Cortex (PMC) is obtained from RMC by undergoing a stir-frying process. Both of them are indicated for different pharmacodynamic action in traditional Chinese medicine, and they have been used in China and other Asian countries for thousands of years. Objective: To establish a method to study the RMC and PMC, revealing their different chemical composition by fingerprint, qualitative, and quantitative ways. Materials and Methods: High-performance liquid chromatography coupled with diode array detector and electrospray mass spectrometry (HPLC-DAD-ESIMS) were used for the analysis. Therefore, the analytes were separated on an Ultimate TM XB-C18 analytical column (250 mm × 4.6 mm, 5.0 μm) with a gradient elution program by a mobile phase consisting of acetonitrile and 0.1% (v/v) formic acid water solution. The flow rate, injection volume, detection wavelength, and column temperature were set at 1.0 mL/min, 10 μL, 254 nm, and 30°C, respectively. Besides, principal components analysis and the test of significance were applied in data analysis. Results: The results clearly showed a significant difference among RMC and PMC, indicating the significant changes in their chemical compositions before and after the stir-frying process. Conclusion: The HPLC-DAD-ESIMS coupled with chemometrics analysis could be used for comprehensive quality evaluation of raw and processed Moutan Cortex.