In the biotechnology industry, highly sensitive and accurate methods are required for monitoring glycosylation of therapeutic recombinant monoclonal antibodies (rMAbs) due to possible effects on bioactivity. At Genentech, a method employing PNGase F digestion, fluorescent labeling of released glycans, and analysis by capillary electrophoresis (CE) is used for routine monitoring of N-linked glycosylation during process development and quality control of therapeutic glycoproteins. In our laboratory, capillary electrophoresis-mass spectrometry (CE-MS) technology was developed to identify minor glycan species in assay and it revealed several unidentified isomeric species. Additional studies indicate that these species (1-10% total glycans) are sample preparation artifacts caused by base-catalyzed epimerization of N-acetylglucosamine (GlcNAc) at the reducing terminus by following the use of commercially available PNGase F and the supplied incubation buffer (pH 7.5). As these isomeric species directly impact the accuracy of the reported results, an optimized PNGase F release step is presented which minimizes and/or eliminates the formation of these artifacts. We have found that PNGase F incubation at pH 5.5 for IgG(1) rMAbs shows no significant decrease in enzyme activity while minimizing GlcNAc epimerization. Implementation of this change has resulted in a more accurate and robust CE-laser-induced fluorescence (LIF) assay and is generally applicable to any analysis requiring PNGase F digestion of rMAbs.