HDL-mediated cholesterol efflux capacity (HDL-CEC) is a functional attribute that may have a protective role in atherogenesis. However, the estimation of HDL-CEC is based on in vitro cell assays that are laborious and hamper large-scale phenotyping.Here, we present a cost-effective high-throughput nuclear magnetic resonance (NMR) spectroscopy method to estimate HDL-CEC directly from serum. We applied the new method in a population-based study of 7603 individuals including 574 who developed incident coronary heart disease (CHD) during 15 years of follow-up, making this the largest quantitative study for HDL-CEC.As estimated by NMR-spectroscopy, a 1-SD higher HDL-CEC was associated with a lower risk of incident CHD (hazards ratio, 0.86; 95%CI, 0.79-0.93, adjusted for traditional risk factors and HDL-C). These findings are consistent with published associations based on in vitro cell assays.These corroborative large-scale findings provide further support for a potential protective role of HDL-CEC in CHD and substantiate this new method and its future applications.