The administration of protein-based pharmaceuticals can cause the in vivo formation of anti-drug antibodies (ADAs) which may reduce the efficacy of the therapy by binding to the protein drug. An accurate determination of the total and ADA-bound concentrations of the drug gives information on the extent of this immune response and its consequences and may help develop improved therapeutic regimens. We present an absolute quantitative method to differentiate between total, free and ADA-bound drug for recombinant human alpha acid glucosidase (rhGAA) in plasma from patients suffering from Pompe's disease. LC-MS/MS quantification of a signature peptide after trypsin digestion of plasma samples before and after an extraction of the total IgG content of plasma with Protein-G coated beads was used to determine the total and the ADA-bound fractions of rhGAA in samples from Pompe patients after enzyme infusion. The methods for total and ADA-bound rhGAA allow quantitation of the drug in the range of 0.5 to 500 µg/mL using 20 µL of plasma and met the regular bioanalytical validation requirements, both in the absence and presence of high levels of anti-rhGAA antibodies. This demonstrates that the ADA-bound rhGAA fraction can be accurately and precisely determined and is not influenced by sample dilution, repeated freezing and thawing or extended bench-top or frozen storage. In samples from a patient with a reduced response to therapy due to ADAs high ADA-bound concentrations of rhGAA were found, while in the samples from a patient lacking ADAs, no significant ADA-bound concentrations were found. Since Protein G captures the complete IgG content of plasma, including all anti-drug antibodies, the described extraction approach is universally applicable for the quantification of ADA-bound concentrations of all non-IgG-based biopharmaceuticals.