Nuclear magnetic resonance (NMR) spectroscopy is often used for the analysis of metabolites in proteinaceous biological specimens. However, the binding of metabolites to proteins impedes accurate quantitation of total metabolite concentrations by NMR, unless protein binding is disrupted by organic solvent precipitation, which increases variance and may result in the loss of volatile metabolites during post-extraction drying. Here, we present an approach for the inference of total metabolite concentrations from Carr-Purcell-Meiboom-Gill NMR spectra via computation of metabolite and sample-specific factors derived from the individual broadening of spectral peaks due to protein-metabolite binding. The method was validated on both synthetic proteinaceous samples and plasma and urine specimens including a certified reference plasma. Furthermore, results were compared with those obtained for methanol extracts of plasma specimens. In summary, our approach obviates the need for protein precipitation, is easy to use, and allows precise and reliable determination of total metabolite concentrations.