This study introduced the use of cotton in 200µL tips, namely cotton HILIC as a convenient tool for enriching large-scale glycopeptides before MS analysis in this study. The optimal loading buffer for cotton HILIC was investigated by using mouse brain as a complex test sample. Subsequently, the performance of cotton HILIC was evaluated by comparing with other two commercial enrichment approaches, venusil HILIC and Oasis MAX in both mouse brain and seminal plasma. Moreover, capacity and recovery rate were evaluated using different milligram of cotton in 200µL tips. Thus, a simple, convenient, and cost-friendly cotton HILIC method was proposed to achieve highly selective intact glycopeptide profiling.