Acid alpha-glucosidase (GAA) is a lysosomal glycogen-catabolizing enzyme, a deficiency in which leads to Pompe disease. Pompe disease can be treated with systemic recombinant human GAA (rhGAA) enzyme replacement therapy (ERT), but the current standard of care has poor uptake in skeletal muscles, limiting clinical efficacy. Further, it is unclear how the specific cellular processing steps of GAA post-delivery to lysosomes impact efficacy. GAA undergoes both proteolytic cleavage and glycan trimming within the endolysosomal pathway, yielding a more active enzyme for hydrolyzing its natural substrate glycogen. The relative contributions for each of these processing steps for increasing rhGAA glycogen hydrolytic activity are unclear. Here, we developed a tool kit of modified rhGAAs that allowed us to dissect the individual contributions of glycan trimming and proteolysis on maturation-associated increases in hydrolytic activity on glycogen. Chemical modifications of terminal sialic acids on N-glycans blocked sialidase activity in vitro and in cellulo, thereby preventing downstream glycan trimming without affecting proteolysis. This sialidase-resistant rhGAA displayed only partial activation following endolysosomal processing, as evidenced by lower catalytic efficiency on glycogen. We also generated enzymatically deglycosylated rhGAA that was shown to be partially activated despite not undergoing proteolytic processing. Taken together, these data suggest that an optimal rhGAA ERT would require both N-glycan and proteolytic processing to attain the most efficient enzyme kinetics for glycogen hydrolysis.