OBJECTIVES The aim of this study was to evaluate the diagnostic utility of plasma metabolomic biomarkers for in-stent restenosis (ISR). BACKGROUND ISR remains an issue for patients after percutaneous coronary intervention. Identification of biomarkers to predict ISR could be invaluable for patient care. METHODS Next-generation metabolomic profiling was performed in the discovery phase from the plasma of 400 patients undergoing percutaneous coronary intervention. In the validation phase, targeted analysis was conducted using stable isotope dilution-multiple reaction monitoring mass spectrometry in another independent group of 500 participants. RESULTS A set of 6 plasma metabolites was discovered and validated for the diagnosis of ISR as early as 1 month after percutaneous coronary intervention. This biomarker panel classified patients with ISR and control subjects with sensitivity of 91% and specificity of 90% in the discovery phase. The diagnostic accuracy in the independent validation phase was 90% (95% confidence interval:87% to 100%). The defined 6 metabolites all belong to sphingolipid and phospholipid metabolism, including phosphatidylcholine diacyl C36:0, phosphatidylcholine diacyl C34:2, phosphatidylinositol diacyl C36:4, phosphatidic acid C34:1, ceramide, and sphingomyelin diacyl 18:1/20:1. These biomarkers play essential roles in cell signaling that regulates the proliferation and migration of vascular smooth muscle cells. CONCLUSIONS Next-generation metabolomics demonstrates powerful diagnostic value in estimating ISR-related metabolic disturbance. The defined plasma biomarkers provide better early diagnostic value compared with conventional imaging techniques. (C) 2017 by the American College of Cardiology Foundation.