•Plant core-fucosylated glycans may elicit an immune response in humans.•Rice cell-derived rhGAAs were produced under eight different culture conditions.•N-glycans of eight rhGAAs were analyzed using UPLC and LC-ESI-HCD-MS/MS.•15 N-glycans were characterized as core-, de-core-, and non-core-fucosylated glycans.•The lowest core-fucosylated condition of rhGAA was 2-FF combined with PF-68. Recombinant human acid alpha-glucosidase (rhGAA) from Chinese hamster ovary cells is the only approved treatment for patients with Pompe disease. In this study, rhGAAs were produced in transgenic rice cell suspension cultures under eight different conditions; untreated, 5 μM of 2-fluoro-l-fucose (2-FF), 50 μM of 2-FF, 100 μM of 2-FF, 100 μM of 2-FF + 0.5% Pluronic F-68 (PF-68), 100 μM of 2-FF + 0.05% Tween 20 (Tw 20), 0.5% PF-68, and 0.05% Tw 20. The N-glycans of eight rhGAAs were analyzed using ultra-performance liquid chromatography (UPLC) and tandem mass spectrometry. The relative quantity (%) of each glycan was obtained from the corresponding UPLC peak area per the sum (100%) of individual UPLC peak area. Fifteen N-glycans, comprising seven core-fucosylated glycans (71.5%, sum of each relative quantities) that have immunogenicity-inducing potential, three de-core-fucosylated glycans (15.4%), and five non-core-fucosylated glycans (13.1%), were characterized with high mass accuracy and glycan-generated fragment ions. The increases or decreases of relative quantities of each glycan from seven rhGAAs were compared with those of untreated control. The percentages of the sum of the relative quantities of core-fucosylated glycans divided by the sums of those of de-core- (core-fucose removed) and non-core-fucosylated glycans were calculated, and the lowest percentage was obtained in 100 μM of 2-FF combined with 0.5% PF-68. These results indicate that the relative quantity of each glycan of rhGAA produced in rice cell suspension cultures is significantly affected by their culture condition. This study performed the comparison of the N-glycan profiles of rice cell-derived rhGAA to identify the core-fucosylated glycans using UPLC and tandem mass spectrometry.