We describe a label‐free relative quantification LC‐MS/MS method for core‐fucosylation in alpha‐2‐macroglobulin (A2MG) immunoprecipitated from human sera. The method utilizes endoglycosidase F partial deglycosylation to reduce glycosylation microheterogeneity, while retaining the innermost N‐acetylglucosamine (GlcNAc) and core fucose. Precursor ion peak areas of partially deglycosylated peptides were obtained and site‐specific core‐fucosylation ratios based on the peak areas of core‐fucosylated and nonfucosylated counterparts were calculated and evaluated for assay development. This assay was applied in a preliminary study of sera samples from normal controls and patients with pancreatic diseases, including pancreatic cancer and chronic pancreatitis. A2MG fucosylation levels at sites N396 and N1424 were found to decrease in both chronic pancreatitis and pancreatic cancer compared to normal controls. The two sites were identified by two peptides and their core‐fucosylation ratios were found to be internally consistent. This method provides a platform to quantify fucosylation levels and can be used to study site‐specific core‐fucosylation aberrations in other glycoproteins for other diseases.