Nuclear magnetic resonance (NMR)-based fluxomics seeks to measure the incorporation of isotope labels in selected metabolites to follow kinetically the synthesis of the latter. It can however equally be used to understand the biosynthetic origin of the same metabolites. We investigate here different NMR approaches to optimize such experiments in terms of resolution and time requirement. Using the isoleucine biosynthesis as an example, we explore the use of different field strengths ranging from 500 MHz to 1.1 GHz. Because of the different field dependence of chemical shift and heteronuclear J couplings, the spectra change at different field strengths. We equally explore the approach to silence the leucine/valine methyl signals through the use of a suitable deuterated precursor, thereby allowing selective observation of the Ile C-13 labeling pattern. Combining both approaches, we arrive at an efficient procedure for the NMR-based exploration of Ile biosynthesis.