In the past few years there has been a large increase in the reporting of the use of dried blood spots (DBS) in drug development. Most of these reports pertain to the technological improvements that have allowed for drug concentration measurements from microliter volumes of sample, issues concerning method development, and exploration of the technique, into other areas such as measurement of macromolecules and metabolite identification. One area that has received less attention and is the subject of this commentary concerns the pharmacokinetic issues that arise from using DBS as opposed to plasma, the mainstay matrix. Measurements of drug concentrations from either plasma or dbs are almost always the sum of bound and unbound drug, but it is the unbound drug in plasma (plasma water) that is the relevant driver of essentially all pharmacokinetic and pharmacodynamic events. Therefore, the critical assumption made is constancy in fraction unbound for plasma, and additionally for blood, constancy of hematocrit and blood cell affinity. Often these assumptions are reasonable and either matrix suffices, but not always. Then the value of one matrix over the other depends on the magnitude of the blood-to-plasma concentration ratio of drug, its clearance and the cause of the deviation from constancy. Additional considerations are the kinetics of distribution within blood and those arising when the objective is assessment or comparison of bioavailability. Most of these issues can be explored and addressed in vitro prior to the main drug development program.