ObjectivesDespite reports highlighting citrate association with different diseases, serum citrate is scarcely used for diagnosis. Existing methods to quantify citrate are limited by their complexity and practicality of implementation. A simple and rapid NMR-based method to measure circulating citrate is described here, and its analytical performance evaluated. Designand Methods: Citrate was quantified from NMR spectra using a non-negative linear least squares deconvolution algorithm. The analytical characteristics of the assay were evaluated using CLSI guidelines. To determine if the assay has adequate sensitivity to measure clinically relevant concentrations of citrate, the assay was used to quantify citrate in apparently healthy adults (n = 553), and in the general population (n = 133,576). ResultsThe LOQ for the assay was determined to be 1.48 mg/dL. Linearity was demonstrated over a wide range of concentrations (1.40–4.46 mg/dL). Coefficients of variation (%CV) for intra- and inter-assay precision ranged from 5.8–9.3 and 5.2–9.6%, respectively. Substances tested did not elicit interference with assay results. Specimen type comparison revealed <1% bias between serum and plasma samples, except for heparin plasma (3% bias). Stability was demonstrated up to 8 days at room temperature and longer at lower temperatures. In a cohort of apparently healthy adults, the reference interval was <1.48–2.97 mg/dL. Slightly higher values were observed in the general population. ConclusionsThe newly developed NMR-based assay exhibits analytical characteristics that allow the accurate quantification of clinically relevant citrate concentrations. The assay provides a simple and fast means to analyze samples for research and clinical studies.