AbstractAlpha‐1,6 core fucosylation (CF) is a unique glycoform of N‐glycans, and studies showed that CF modifications are involved in the occurrence and progression of various diseases and may provide potential disease biomarkers. Current strategies for the CF glycoproteome are often based on multistep enrichment of glycoproteins or glycopeptides and sequential cleavage with different glycosidases to truncate the N‐glycans. Although the detection ability of low‐abundance glycoproteins is improved, sample loss, high cost, and the time‐consuming multistep operation also affect the reproducibility of results and the practicality of the method. Here we developed a single‐step truncation (SST) strategy and evaluated its potential for the CF glycoproteome of human serum. The SST strategy has the advantages of fewer operational steps, lower cost, higher number of identifications, and better quantitative stability compared with previous approaches and provides an efficient solution for large‐scale quantitative analysis of the CF glycoproteome. © 2024 Wiley Periodicals LLC.Basic Protocol 1: Single‐step truncation strategy for core fucosylation glycoproteome analysis in human serumBasic Protocol 2: Liquid chromatography–tandem mass spectrometry quantification of site‐specific core fucosylation glycopeptidesAlternate Protocol: Pretreatment of cellular samples of core fucosylation glycoproteome with single‐step truncation strategy