Aberrant glycosylation plays a pivotal role in a diverse set of diseases, including cancer. A microfluidic lectin blotting platform is introduced to enable and expedite the identification of protein glycosylation based on protein size and affinity for specific lectins. The integrated multistage assay eliminates manual intervention steps required for slab-gel lectin blotting, increases total assay throughput, limits reagent and sample consumption, and is completed using one instrument. The assay comprises non-reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by online post-sizing SDS filtration and lectin-based affinity blotting. Important functionality is conferred through both device and assay advances that enable integration of nanoporous membranes flanking a central microchamber to create sub-nanoliter volume compartments that trap SDS-protein complexes and allow electrophoretic SDS removal with buffer exchange. Recapitulation of protein binding for lectin was optimized through quantitative assessment of SDS-treated green fluorescent protein. Immunoglobulin A1 aberrantly glycosylated with galactose-deficient O-glycans was probed in ∼6 min using ∼3 μL of sample. This new microfluidic lectin blotting platform provides a rapid and automated assay for the assessment of aberrant glycosylation. © 2011 American Chemical Society.