Follicular fluid, an important microenvironment for the development of oocytes, contains many proteins that are glycosylated with N-linked glycans. This study aimed 1) to present an initial analysis of the N-linked glycan profile of bovine follicular fluid using hydrophilic interaction liquid chromatography, anion exchange chromatography, HPLC (high pressure liquid chromatography)-based separations and subsequent LC-MS/MS (liquid chromatography - mass spectrometry/mass spectrometry) analysis; 2) to determine differences in the N-glycan profile between follicular fluid from dominant and subordinate follicles from dairy heifers and lactating dairy cows; and 3) to identify alterations of the N-glycan profile of follicular fluid during preovulatory follicle development using newly selected, differentiated (preovulatory), and luteinized dominant follicles from dairy heifers and lactating cows. We found that the majority of glycans on bovine follicular fluid are based on biantennary hypersialylated structures; where glycans are sialylated on both the galactose (Gal) and n-acetylglucosamine (GlcNAc) terminal sugars. A comparison of follicular fluid N-glycans from cows and heifers indicated higher levels of nonsialylated glycans with a lower proportion of sialylated glycans in cows than heifers. Overall, as the follicle develops from selection, differentiation, and luteinization in both cows and heifers there is an overall decrease in sialylated structures on follicular fluid N-glycans.