PurposeTo demonstrate the framework of a novel Hadamard-encoded spectral editing approach for simultaneously detecting multiple low-concentration brain metabolites in vivo at 3T. MethodsHERCULES (Hadamard Editing Resolves Chemicals Using Linear-combination Estimation of Spectra) is a four-step Hadamard-encoded editing scheme. 20-ms editing pulses are applied at: (A) 4.58 and 1.9 ppm; (B) 4.18 and 1.9 ppm; (C) 4.58 ppm; and (D) 4.18 ppm. Edited signals from γ-aminobutyric acid (GABA), glutathione (GSH), ascorbate (Asc), N-acetylaspartate (NAA), N-acetylaspartylglutamate (NAAG), aspartate (Asp), lactate (Lac), and likely 2-hydroxyglutarate (2-HG) are separated with reduced signal overlap into distinct Hadamard combinations: (A+B+C+D); (A+B–C–D); and (A–B+C–D). HERCULES uses a novel multiplexed linear-combination modeling approach, fitting all three Hadamard combinations at the same time, maximizing the amount of information used for model parameter estimation, in order to quantify the levels of these compounds. Fitting also allows estimation of the levels of total choline (tCho), myo-inositol (Ins), glutamate (Glu), and glutamine (Gln). Quantitative HERCULES results were compared between two grey- and white-matter-rich brain regions (11 min acquisition time each) in 10 healthy volunteers. Coefficients of variation (CV) of quantified measurements from the HERCULES fitting approach were compared against those from a single-spectrum fitting approach, and against estimates from short-TE PRESS data. ResultsHERCULES successfully segregates overlapping resonances into separate Hadamard combinations, allowing for the estimation of levels of seven coupled metabolites that would usually require a single 11-min editing experiment each. Metabolite levels and CVs agree well with published values. CVs of quantified measurements from the multiplexed HERCULES fitting approach outperform single-spectrum fitting and short-TE PRESS for most of the edited metabolites, performing only slightly to moderately worse than the fitting method that gives the lowest CVs for tCho, NAA, NAAG, and Asp. ConclusionHERCULES is a new experimental approach with the potential for simultaneous editing and multiplexed fitting of up to seven coupled low-concentration and six high-concentration metabolites within a single 11-min acquisition at 3T.