Dried blood spots (DBS) are widely used as a non-invasive sampling method, especially in newborn screening (NBS). Despite its numerous advantages, conventional DBS might be limited by the hematocrit effect when analyzing a punch, depending on its position in the blood spot. This effect could be avoided using hematocrit-independent sampling devices such as the hemaPEN®. This device collects blood through integrated microcapillaries, and a fixed blood volume is deposited on a pre-punched paper disc. NBS programs are increasingly poised to include lysosomal disorders, given the availability of treatments that improve clinical outcomes if detected early. In this study, the effect of hematocrit and punch position in the DBS on the assay of 6 lysosomal enzymes was evaluated on 3 mm discs pre-punched in hemaPEN® devices compared to 3 mm punches from the PerkinElmer 226 DBS.