Abstract: To study the multivariate statistical analysis and metabolic regulation mechanism of rhubarb in the treatment of blood stasis syndrome through the ultra-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry (UHPLC-Q-TOF/MS). In this study, multivariate statistical analysis was used to comprehensively analyze the differential characteristics of metabolites with significant regulatory effect in plasma samples of blood stasis model rats, analyze the changes of differential metabolites of rhubarb regulating blood stasis syndrome, and analyze the pathway enrichment of identified metabolites. The results showed that the differentiation of plasma differential metabolites between the model group and the control group was good (Q² > 0.5), and 44 differential metabolites all had different degrees of callback. The expressions of PC(15:0/20:2(11Z,14Z)), PC(18:1(9Z)/18:1(9Z)), salicylaldehyde and glycolic acid were down-regulated in the rat model of blood stasis syndrome, and rhubarb could up-regulate them. The expression levels of (±)8-HETE, taurodeoxycholic acid and γ-murocholic acid were up-regulated in the blood stasis rat model, which could be down-regulated by rhubarb. Differential metabolites are enriched in 97 metabolic pathways, involving lipid metabolism pathways, inflammatory factors and immune pathways, and steroid hormone synthesis pathways. This study clarified the mechanism of rhubarb in the treatment of blood stasis syndrome from the perspective of plasma metabolism, and provided a theoretical basis for the further development and clinical application of rhubarb. This study has been approved by the experimental animal Ethics Committee of Shaanxi University of Traditional Chinese Medicine (No. SUCMDL202103009002).