Dried bloodspot (DBS) technology has been available for many decades but only in the last five years has it been considered for routine bioanalysis of blood samples collected on preclinical and clinical studies as part of a drug development programme. Advantages of using DBS versus typical plasma samples include smaller blood volumes, less processing of the samples (e.g. no centrifugation) and no requirement for storing or shipping of the samples at frozen temperatures. The current study compared blood concentrations (AUC(0-t) and C(max)) from rats given an oral dose of acetaminophen (APAP) using two different sampling sites (caudal venepuncture versus tail snip), two different collection methods (3 separate 15 μL ethylenediaminetetraacetic acid [EDTA]-coated capillary tubes versus an EDTA integrated capillary blood collection system) and variability between blood spots on one card. There were no noteworthy differences (i.e. two-fold or greater) in blood concentrations of APAP using the different sites or methods. Furthermore, comparisons of the APAP blood concentrations in the original spot to a duplicate bloodspot from the same bloodspot card were within 12% of the original concentration.