The glycosylation of biologics is an important factor in pharmacological functions such as efficacy, safety, and biological activity and is easily affected by subtle changes in the cellular environment. Hence, comprehensive and in-depth glycomic characterization of biological products should be performed to ensure product quality and process consistency prior to regulatory approval, but it is still highly challenging due to glycan microheterogeneity produced by enzymatic machinery. In this study, we have developed a systematic methodology for the separation and characterization of various glycans of biotherapeutics using the combination of solid-phase extraction (SPE) and high resolution LC/MS. Neutral and multiple-acidic glycans were selectively fractionated by SPE with a porous graphitized carbon (PGC) cartridge according to their molecule size and polarity (acidity, p Ka). Subsequent LC-MS and -MS/MS analyses enabled us to obtain glycan compositions, structures, and quantitative information. Indeed, we have successfully performed glycomic characterization of agalsidase-beta, a representative therapeutic enzyme containing both phosphorylated and sialylated glycans. In addition, a comparative analysis of functional glycans released from different batches of enzymes was performed to verify our method. These results suggest that stepwise PGC-SPE and LC/MS/MS pairwise assays can be used as an efficient tool to detect glycosylation changes of therapeutic glycoproteins including abundant acidic species in biologics or biosimilar development.