Background: There is considerable interest in the determination of relative abundances of human metabolites in plasma (and potentially excreta) with reasonable accuracy early on in the drug development process in order to make scientifically sound decisions with regard to the presence of potentially active or toxic disproportionate metabolites. At this point, authentic metabolite standards are generally not available. Results: A new methodology is proposed for the estimation of metabolite concentrations in the absence of authentic standards. A reference sample containing radiolabeled metabolites of interest is produced by incubating the ¹⁴C-labeled drug in vitro, and mixed with a sample to be quantitated containing the unlabeled metabolites. The ¹²C/¹⁴C isotope ratio is measured with high-resolution ESI–MS for each metabolite, and used as a basis for quantitation of the cold metabolite based on the concentration of radioactive metabolite, determined from independent analysis of the radioactive sample with LC-radiochemical detection. The ¹⁴C-labeled metabolite serves as an isotopically labeled internal standard, which corrects for any variations in injection volume, sample preparation, MS intensity drift, matrix effects and/or saturation of electrospray ionization. The approach was validated by the analysis of solutions containing variable amounts of the analyte with a fixed amount of radioactive standard on a QToF Synapt^® G2 MS system. The same methodology was also successfully applied to first-in-human plasma samples analyzed on a LTQ-Orbitrap^®. Conclusion: The metabolite abundances obtained by ¹²C/¹⁴C isotope ratio measurements showed suitable accuracy and precision and were very close to those obtained with matrix mixing. The parent drug concentrations also corresponded well with the bioanalytical results obtained with a validated LC–MS/MS method.