Assessment of α-galactosidase A (α-GAL) activity is a reliable method for evaluating the pharmacological efficacy of migalastat (Galafold) in Fabry disease patients undergoing chaperone therapy. Although some of mutant α-GAL retain normal catalytic function, their aberrant conformations lead to intracellular degradation and impaired lysosomal trafficking. Approximately 30% of Japanese Fabry patients are reported to be responsive to migalastat. In such cases, measurable enzyme activity following migalastat administration suggests successful stabilization and lysosomal delivery of the mutant enzymes. To monitor long-term therapeutic effects, dried blood spots (DBSs) are periodically prepared from patient samples as a convenient method for α-GAL activity measurement. However, residual migalastat in blood escaped from excretion can competitively inhibit the substrate 4-Metylumbellifel-α-D-galactoside (4-MUG) during DBS-based assays, resulting in underestimated enzyme activity. To address this, we investigated the impact of migalastat interference and demonstrated that removing migalastat using Amicon®Ultra filter from DBS samples enables accurate assessment of α-GAL activity for monitoring therapeutic response in Fabry disease.