The contribution of a 13C-enriched substrate to the acetyl-CoA pool in animal tissues is typically measured by analysis of glutamate enrichment from tissue extracts. 13C 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 1H NMR measurement of glutamate C4 enrichment from tissue extracts. The method is demonstrated with heart and hindlimb muscle tissue extracts of rats infused with [2,4,6,8-13C4]-octanoate, a source of [2-13C]acetyl-CoA. Glutamate C4 enrichment in extracts of individual hindlimb soleus muscles weighing approximately 150 mg and containing approximately 0.3 mumol of glutamate was quantified by 1H NMR within about 40 min. Glutamate C4 enrichment measurements by 1H NMR in heart and gastrocnemius muscle were also highly correlated with independent measurements obtained from 13C NMR isotopomer analysis.