The processing of traditional Chinese medicine (TCM) is a necessary practice and usually occurs before most herbs are prescribed. According to Chinese medicine theory, raw (RDR) and stir-frying processed (PDR) Drynariae Rhizoma have different clinical applications. The purpose of this study was to establish HPLC fingerprints coupled with chemometric methods to evaluate the differences between RDR and PDR. Multivariate chemometric methods were applied to analyze the obtained HPLC fingerprints, including hierarchical cluster analysis (HCA), principle components analysis (PCA), and partial least squares discriminant analysis (PLS-DA). The results indicated that RDR and PDR samples showed a clear classification of the two groups, and seven chemical markers having great contributions to the differentiation were screened out. The findings suggested that 5-hydroxymethyl-2-furaldehyde (5-HMF) with a variable importance in the project (VIP > 1) can be used to differentiate between RDR and PDR. Moreover, 5-HMF, naringin, and neoeriocitrin were determined to evaluate their contents in RDR and PDR. The chemometrics combined with the quantitative analysis based on HPLC fingerprint results indicated that stir-frying processing may change the contents and types of components in Drynariae Rhizoma. These changes are probably responsible for the various pharmacological effects of RDR and PDR.