The contribution of a¹³C-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 direct¹H 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 ≈150 mg and containing approximately 0.3 μmol of glutamate was quantified by¹H NMR within about 40 min. Glutamate C4 enrichment measurements by¹H NMR in heart and gastrocnemius muscle were also highly correlated with independent measurements obtained from¹³C NMR isotopomer analysis.