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Background: Mahuang decoction (MHD), a famous classic traditional Chinese formula, has been extensively applied for treating cold, influenza, asthma, acute bronchitis, and other pulmonary diseases. However, the interaction among four drugs of MHD has not been clearly deciphered from the aspect of molecular composition. Objective: To assess the quality of MHD and explore the interplay among different prescription drugs. Materials and Methods: A reversed-phase high performance liquid chromatography (RP-HPLC) coupled with diode array detector (DAD) method for the simultaneous separation and determination of nine bioactive components was developed. A somatomedin A (SMA)-phenyl column (4.6 mm × 250 mm, 5 μm) was eluted by a gradient mobile phase contained acetonitrile and 0.05% formic acid-0.05% triethylamine aqueous solution. Four detection wavelengths (210, 252, 278, and 291 nm) were utilized for the quantitative analysis due to the different ultraviolet (UV) spectra of these compounds. Results: Satisfactory separation was obtained for all the components, and the assay was fully validated in respects of linearity, precision, stability, and accuracy. It was found that the calibration curves for all analytes showed good linearity (R2≥ 0.9991) within the test ranges. The relative standard deviations (RSDs) for intra-.and interday repeatability were not more than 1.70 and 2.66%, respectively. The spike recoveries of nine components varied from 97.50 ± 1.69 to 99.27 ± 1.37%. Conclusion: The established method was successfully applied to analyze nine active compounds in decoction samples of various drug compatibilities of MHD. The variations of contents were obvious for different combinations, which hinted the mutual promotion or inhibition of componential dissolution among four herbs of MHD. [ABSTRACT FROM AUTHOR]