Isotope dilution mass spectrometry (ID-MS) has been used as a primary method for the absolute quantitative determination. Although the conventional methods in quantitative proteomics, such as the absolute (e.g., synthetic proteins or peptides) or relative quantifications (e.g., ICAT, mTRAQ), have been considered as a reliable approach in shotgun proteomics, these methods have unfortunately several bottlenecks such as imperfect labeling, and time consumption of labeling for proteome samples. To improve above-mentioned problems, we applied the carbamidomethylation-based isotope labeling (iCCM) for absolute quantification of human growth hormone (hGH) and brain natriuretic peptide-32 (BNP-32) in human plasma in this study. In order to evaluate the iCCM labeling for absolute quantification, plasma sample was first labeled with iodoacetamide (IAA) as carbamidomethylation (CM), while both hGH and BNP-32 standards were isotopically alkylated with IAA isotope (IAA-13C2D2) as iCCM, followed by spiking those iCCM-labeled standards to plasma sample labeled with CM. The resulting plasma sample was directly performed tryptic digestion and subsequently applied to nLC-ESI-MS/MS analysis so as to obtain the absolute amounts of both hGH and BNP-32. In conclusion, iCCM-based ID-MS provides the simple, rapid, and high reproductive method for the assessment of the ratio of CM/iCCM, resulting in absolute quantification of hGH and BNP-32.