The contribution of a 1 3 C-enriched substrate to the acetyl-CoA pool in animal tissues is typically measured by analysis of glutamate enrichment from tissue extracts. 1 3 C NMR analysis offers the advantages of minimal sample processing and high information content, but has a low analytical sensitivity compared to other methods of tracer analysis such as GC/MS. We present a sensitive, simple, and direct 1 H NMR measurement of glutamate C4 enrichment from tissue extracts. The method is demonstrated with heart and hindlimb muscle tissue extracts of rats infused with [2,4,6,8- 1 3 C 4 ]octanoate, a source of [2- 1 3 C]acetyl-CoA. Glutamate C4 enrichment in extracts of individual hindlimb soleus muscles weighing ~150 mg and containing approximately 0.3 μmol of glutamate was quantified by 1 H NMR within about 40 min. Glutamate C4 enrichment measurements by 1 H NMR in heart and gastrocnemius muscle were also highly correlated with independent measurements obtained from 1 3 C NMR isotopomer analysis.