Background The curative effect of Cuscutae Semen could be adjusted after stir-frying, and it’s difficult to distinguish between the raw Cuscutae Semen (CS) and stir-frying processed Cuscutae Semen (SFCS) in clinical practice. Objective The paper aims to establish the fingerprints of raw and fried Cuscutae Semen products by high performance liquid chromatography (HPLC) so that could analyze the compounds in raw and stir fried products qualitatively and quantitatively. At the same time, the chemometrics method was used to evaluate the difference between CS and SFCS to provide reference for the research of traditional Chinese medicine Cuscutae Semen. Method The raw and stir-frying processed Cuscutae Semen products related substances were separated on C18 column (250 mm × 4.6 mm, 5 µm) maintained at 30 °C (column temperature). The mobile phase comprised 0.1% formic acid aqueous solution (A) and acetonitrile (B), and a detection wavelength of 328 nm. The datas were imported into statistical software for chemometric analysis. Results The developed HPLC method exhibits good linearity and has good systematic applicability. The content of these 12 compounds in the samples was further determined and the data analysed by chemometrics. The results showed that the composition of CS changed on heating and HCA showed that CS and SFCS could be clearly distinguished. PCA showed that six components caused differences, namely neochlorogenic acid, cryptophyllogenic acid, caffeic acid, quercetin, isorhamnetin and kaempferol. Conclusions This study unequivocally establishes a chromatographic fingerprint method intended for the extensive analysis of raw and stir-frying processed CS, which could substantially enhance the quality control of Cuscutae Semen and the rational development and utilization of traditional Chinese medicine (TCMs) resources. Highlights This method for the simultaneous quantification of multiple compounds in CS and SFCS, and revealing the components responsible for the differences between raw and processed products. This will provide support for quality control of this herbal medicine.