Although many isobaric labeling-based targeted quantification methods by mass spectrometry (MS) have been developed and improved to meet proteome research requirements, some disadvantages limit their applications. For instance, some methods require expensive reagents and others have slight differences in chromatographic retention times due to the deuterium isotope effect when dimethylation labeling was performed with binary isotopic reagents of formaldehyde (d0 and d2). Therefore, a novel absolute quantitation method for a target proteome using ¹⁸O/metal isobaric labeling of a quantification concatamer protein (QconCAT) as the internal standard combined with multiple reaction monitoring mass spectrometry (MRM MS) was established. The experimental results showed that the peptides can be very efficiently labeled with both ¹⁸O + Ho and ¹⁶O + Tm, and that the labeled peptides remained stable for 7 days. In addition, the labeled peptides with ¹⁸O + Ho and ¹⁶O + Tm displayed nearly identical behaviors in high-performance liquid chromatographic and mass spectrometric analyses. Furthermore, using a tryptic digest of a complex biological sample as a matrix, the linear range spanned two orders of magnitude, the relative errors of standard samples were less than 20% and the LOQ was 0.7 fmol μL⁻¹ (RSD < 10%) when the method was evaluated with two transitions of a randomly selected peptide (TATVDDIDNIYR) from a protein of a Thermoanaerobacter tengcongensis sample as a model. For further evaluation of our method, the absolute quantitation of 20 drug metabolic enzymes in 5 human liver microsomes was performed using the established method, and the results are consistent with those reported in the literature. These results suggest that this method using ¹⁸O/metal isobaric labeling combined with MRM MS can be used as a new approach for the absolute quantitation of a targeted proteome in complex biological samples.