Nuclear magnetic resonance (NMR) spectroscopy is widely used in metabolomics to perform quantitative profiling of low-molecular weight compounds from biological specimens. The measurement of endogenous metabolites using NMR has proven to be a powerful tool to identify new metabolic biomarkers in physiological and pathological conditions, and to study and evaluate treatment efficiency. In this study we present a rapid approach to indirectly quantify 13C enriched molecules using one-dimensional (1D) 1H NMR. We demonstrate this approach using isotopically labeled [1,6-13C]glucose and in four different cell lines. We confirm the applicability of this approach for treatment follow-up, utilizing a renal cancer cell line with rapamycin as a tool compound to study changes in metabolic profiles. Finally, we validate the applicability of this method to study metabolic biomarkers from ex vivo tumor extracts, after infusion, using isotopically enriched glucose. Given the high throughput and increased sensitivity of direct-detect 1H NMR, this analytical approach provides an avenue for simple and rapid metabolic analysis of biological samples including blood, urine, and biopsies.