Unambiguous identification of individual metabolites present in complex mixtures such as biofluids constitutes a crucial prerequisite for quantitative metabolomics, toward better understanding of biochemical processes in living systems. Increasing the dimensionality of a given NMR correlation experiment is the natural solution for resolving spectral overlap. However, in the context of metabolites, natural abundance acquisition of (1)H and (13)C NMR data virtually excludes the use of higher dimensional NMR experiments (3D, 4D, etc.) that would require unrealistically long acquisition times. Here, we introduce projection NMR techniques for studies of complex mixtures, and we show how discrete sets of projection spectra from higher dimensional NMR experiments are obtained in a reasonable time frame, in order to capture essential information necessary to resolve assignment ambiguities caused by signal overlap in conventional 2D NMR spectra. We determine optimal projection angles where given metabolite resonances will have the least overlap, to obtain distinct metabolite assignment in complex mixtures. The method is demonstrated for a model mixture composition made of ornithine, putrescine and arginine for which acquisition of a single 2D projection of a 3D (1)H-(13)C TOCSY-HSQC spectrum allows to disentangle the metabolite signals and to access to complete profiling of this model mixture in the targeted 2D projection plane. Copyright (C) 2010 John Wiley & Sons, Ltd.