Our laboratory has focused on the re-engineered of the secretory pathway of Pichia pastoris to perform glycosylation reactions that mimic processing of N-glycans in humans and other higher mammals (1,2). A reporter protein with a single N-linked glycosylation site, a His-tagged Kringle 3 domain of human plasminogen (K3), was used to identify combinations of optimal leader/catalytic domain(s) to recreate human N-glycan processing in the Pichia system. In this chapter we describe detailed protocols for high-throughput purification of K3, enzymatic release of N-glycans, matrix-assisted laser desorption ionization time-of-flight and high-performance liquid chromatography analysis of the released N-glycans. The developed protocols can be adapted to the characterization of N-glycans from any purified protein expressed in P. pastoris.