This study aims to investigate the transfer of key substance at different stages of the preparation process of Prunellae Spica ointment, providing a reference for the establishment of quality standards for Prunellae Spica ointment. Using 15 batches of Prunellae Spica paste benchmark samples as the research objects, a comprehensive component analysis was conducted using UHPLC-Q-Exactive Orbitrap MS technology. The HPLC fingerprint of the reference samples was established, and chemometric analysis was employed to screen the main chemical components responsible for quality differences among different batches of Prunellae Spica ointment benchmark samples. Additionally, by combining component analysis and reference standard comparison, common components were identified, and a method for determining the content of these common components was developed. The transfer rates of these components were also calculated.The results indicated that 86 compounds were identified from Prunellae Spica and extraction solution, fluid extract,ointment. In the fingerprint, 18 common peaks were labeled, and 8 components were identified, including salvianic acid A, protocatechuic acid, caffeic acid, rutin, hyperoside, salviaflaside, rosmarinic acid, and luteolin. Chemometric analysis classified the reference samples into 4 categories, and 8 components contributing to quality differences were screened out. During the transfer process from Prunellae Spica to ointment, the transfer rates of the components were relatively stable except for caffeic acid and luteolin, which exhibited lower average transfer rates and significant losses during the process, indicating poor retention from Prunellae Spica to ointment. This study analyzed the transfer patterns of the reference samples of Prunellae Spica ointment by integrating component analysis, paste yield, fingerprint, chemometric analysis, and multi-component content determination, providing a reference for the quality control and evaluation of Prunellae Spica ointment.