•N-glycans containing O-acetylated SA affect therapeutic glycoprotein efficacy.•O-acetylated glycans of Myozyme for treating Pompe disease have not been reported.•This study investigated O-acetylated glycans on Myozyme using LC-ESI-HCD-MS/MS.•Four types of 17 O-acetylated glycans constitute 6.4% of Myozyme’s total glycans.•Glycopeptide analysis by nano-LC-ESI-HCD-MS/MS confirmed six O-acetylation sites. O-acetylated sialic acid (SA) attached to the N-glycans of therapeutic glycoproteins reportedly inhibit sialidase activity, increase protein half-life, decrease protein antigenicity, and stabilize protein conformation. Recombinant human acid α-glucosidase (Myozyme) is the only drug approved by the United States Food and Drug Administration for the treatment of Pompe disease. In this study, unreported N-glycans containing O-acetylated SA in Myozyme and the relative quantities of total glycans were investigated using liquid chromatography (LC)-electrospray ionization (ESI)-high-energy collisional dissociation (HCD) tandem mass spectrometry (MS/MS). The 17 N-glycans (6.4% of total glycans) containing mono-, di-, mono/di-, and di/di-O-acetylated N-acetylneuraminic acid (Neu5Ac) were identified with mass accuracy, glycan-generated fragment ions, and the retention time on an LC column. The analysis of peptides containing mono- and/or di-O-acetylated Neu5Ac ions sorted from all peptides using nano-LC-ESI-HCD-MS/MS confirmed six O-acetylation sites (Asn 140, Asn 233, Asn 390, Asn 470, Asn 652, and Asn 882), at least five of which (Asn 140, Asn 233, Asn 390, Asn 470, and Asn 652) could contribute to the drug efficacy or cellular uptake of Myozyme. This is the first study to identify N-glycans containing O-acetylated Neu5Ac and O-acetylation sites in Myozyme.