N-linked glycosylation is a posttranslational modification affecting protein folding and protein function. The N-linked glycosylation pathway in algae is poorly characterized, and further knowledge is needed to understand the cell biology of algae and the evolution of N-linked glycosylation. This study investigated the N-linked glycosylation pathway in the open ocean diatom Thalassiosira oceanica and identified N-linked glycosylated peptides through solid-phase extraction of N-linked glycosylated peptides (SPEG). We characterized 118 N-linked glycosylated peptides from samples of cells grown in high- and low-iron conditions. The identified peptides had 81% NXT-type motifs, with X being any amino acids except proline. The presence of N-linked glycosylation sites in the iron starvation-induced protein 1a (ISIP1a) confirmed its predicted topology, contributing to the biochemical characterization of ISIP1 proteins.