Lipidomics, a branch of metabonomics, could provide a powerful technique for discovery of lipid molecules to reveal disease status and drug efficacy. The Shenqi pill (SQP) is a representative prescription for clinical application in the prevention and treatment of kidney-yang deficiency syndrome (KYDS). However, its effect mechanism is still not clear. This article aims to reveal the intervention effect of SQP on KYDS from the perspective of lipid metabolism. In this study, SQP was used to intervene in the rat model of KYDS, on the foundation of successfully replicating the rat model of KYDS induced by corticosterone. The MetaboAnalyst tool was used for analysis of the serum metabolic profile and pattern recognition of KYDS model, based on UPLC-SYNAPT-G2-Si-HDMS. Finally, twenty-two potential lipid biomarkers related to the KYDS model were characterized, and the effects of SQP on regulating potential lipid markers in serum of KYDS model were analyzed. There were 10 biomarkers and seven metabolic pathways closely related to SQP therapy for KYDS were found. The action mechanism and targets of SQP in treating KYDS were explored based on high-throughput lipidomics. This work could provide valuable data and scientific evidence in subsequent studies for the treatment of KYDS.