Dried blood spot (DBS) sampling, the collection of whole blood samples on paper, is being evaluated for its use in quantitative analysis. The aim of this investigation was to evaluate the impact of sample hematocrit (HCT) and punch location on assay bias in the measurement of compounds with varying physicochemical properties, when a portion of a DBS sample is analyzed. In addition, the statistical significance of factors such as compound, card type, HCT level, spot position on a card and the location of the punch on the spot were evaluated.