Purpose: To investigate the spectrum-effect relationship between HPLC fingerprints and the inhibitory effect on MUC5AC mucin of Pinelliae Rhizoma Praeparatum Cum Alumine (PRPCA).Methods: The fingerprints of 20 PRPCA batches were established using HPLC and their similarities or differences were analyzed using hierarchical cluster analysis (HCA) and principal component analysis (PCA). The inhibitory effects of MUC5AC mucin were evaluated in LPS-treated NCI-H292 cells. The spectrum-effect relationship between common chromatographic peaks and MUC5AC inhibition was established using a partial least squares-discriminant analysis (PLS-DA).Results: Fifteen common chromatographic peaks were identified by analyzing HPLC fingerprints, with uridine, tyrosine, uracil, and inosine found as possible markers to distinguish the PRPCA from different sources. Spectrum-effect relationship analysis showed that the chromatographic peaks 5, 6, 10 (vernine), 12 (5-hydroxymethylfurfural), 14 (tryptophan) and 15 (adenosine) were closely associated with the inhibitory effect on MUC5AC mucin.Conclusion: The spectrum-effect relationship between HPLC fingerprints and the inhibitory effect on MUC5AC mucin of PRPCA was successfully established in the present study. Our findings further reveal the material basis of PRPCA and provide an effective method for its quality control.