The scientifically and logistically best way of application of the internal standard (IS) in the analysis of dried blood spots (DBS) analysis is still a matter of debate and investigation. Most commonly the IS is added in the solvent used for extraction of the discs punched from DBS. In this case, the recovery of the non-extracted IS is complete while the recovery of the analyte extracted from DBS is different from the IS. An alternative way for addition of the IS was investigated. A homogeneous distribution and absorption of the test compound across the spots was demonstrated by spraying a solution of a radiolabeled test compound (mimicking an IS solution) onto DBS. This spray-on technique is convenient and easily automatable. Spraying of the solution was rapid, precise and reproducible, and therefore seems to be suitable for routine analysis of DBS by offline and online extraction.