Despite quantitative glycomics is premised on highly efficient and reproducible oligosaccharide liberation from human serum glycoproteins, it should be noted that there is no validated protocol of which deglycosylation efficiency is proven to be quantitative. Aiming to establish a standard procedure to release N-glycans present in whole human serum glycoproteins by peptide N-glycosidase F (PNGase F), we evaluated various conditions, where serum glycoproteins were denatured by either reducing agents, surfactants, protease treatment or the combination of these pretreatments prior to PNGase F digestion, in terms of their efficiencies of the liberation of major N-glycans. It was demonstrated that de-N-glycosylation efficiency could differ significantly depending on the condition employed, thus drawing attention for the accumulation and comparison of glycomic data. The maximum de-N-glycosylation was achieved only when serum was subjected to the reductive alkylation in the presence of 2-hydroxyl-3-sulfopropyl dodecanoate, a surfactant employed for solubilizing proteins, or other related analogues, followed by tryptic digestion prior to PNGase F treatment. The optimized de-N-glycosylation protocol permitted relative and absolute quantitation of as many as 23 major N-glycans present in serum glycoproteins for the first time. Furthermore, the process of PNGase F-catalyzed de-N-glycosylation of whole serum glycoproteins was characterized kinetically, which allowed accurate simulation of PNGase F-catalyzed de-N-glycosylation required in clinical glycomics using human serum samples. The present study reveals for the first time the absolute concentrations of major N-glycans existing in human serum whole glycoproteins by means of the optimized protocol for quantitative glycomics.