Glycosylation, as the most prominent posttranslational modification, is recognized as an important quality attribute of monoclonal antibodies affected by various bioprocess parameters and cellular physiology. A method of lectin‐based bio‐layer interferometry (LBLI) to relatively rank galactosylation and fucosylation levels was developed. For this purpose, Fc‐glycosylated immunoglobulin G (IgG) was recombinantly produced with varying bioprocess conditions in 15 L bioreactor and accumulated IgG was harvested. The reliability, the robustness and the applicability of LBLI to different samples has been proven. Data obtained from LC–MS analysis served as reference and were compared to the LBLI results. The introduced method is based on non‐fluidic bio‐layer interferometry (BLI), which becomes recently a standard tool for determining biomolecular interactions in a label‐free, real‐time and high‐throughput manner. For the intended purpose, biotinylated lectins were immobilized on disposable optical fiber streptavidin (SA) biosensor tips. Aleuria aurantia lectin (AAL) was used to detect the core fucose and Ricinus communis agglutinin 120 (RCA120) to determine galactosylation levels. In our case study it could be shown that fucosylation was not affected by variations in glucose feed concentration and cultivation temperature. However, the galactosylation could be correlated with the ratio of mean specific productivity (q _P ) and ammonium (q _NH4+ ) but was unrelated to the ratio of mean q _P and the specific glucose consumption (q _gluc ). This presented method strengthens the applicability of the BLI platform, which already enables measurement of several product related characteristics, such as product quantity as well as kinetic rates (k _d ,k _on ) and affinity constants (k _D ) analysis.