Monoclonal antibodies are typically glycosylated at asparagine residues in the Fc domain, and glycosylation heterogeneity at the Fc sites is well known. This paper presents a method for rapid analysis of glycosylation profile of the therapeutic monoclonal antibody trastuzumab from different production batches using electrospray quadrupole ion-mobility time-of-flight mass spectrometry (ESI-Q-IM-TOF). The global glycosylation profile for each production batch was obtained by a fast LC-MS analysis, and comparisons of the glycoprofiles of trastuzumab from different lots were made based on the deconvoluted intact mass spectra. Furthermore, the heterogeneity at each glycosylation site was characterized at the reduced antibody level and at the isolated glycopeptide level. The glycosylation site and glycan structures were confirmed by performing a time-aligned-parallel fragmentation approach using the unique dual-collision cell design of the instrument and the incorporated ion-mobility separation function. Four different production batches of trastuzumab were analyzed and compared in terms of global glycosylation profiles as well as the heterogeneity at each glycosylation site. The results show that each batch of trastuzumab shares the same types of glycoforms but relative abundance of each glycoforms is varied.