A procedure to map N-glycosylation sites is presented here. It can be applied to purified proteins as well as to highly complex mixtures. The method exploits deglycosylation by PNGase F in a diagonal, reverse-phase chromatographic setup. When applied to 10 microL of mouse serum, affinity-depleted for its three most abundant components, 117 known or predicted sites were mapped in addition to 10 novel sites. Several sites were detected on soluble membrane or receptor components. Our method furthermore senses the nature of glycan structures and can detect differential glycosylation on a given site. These properties--high sensitivity and dependence on glycan imprinting--can be exploited for glycan-biomarker analysis.