Background: Online automated extraction of dried blood spots (DBS) via direct extraction to a solid-phase extraction (SPE) cartridge and bioanalysis by high-resolution accurate mass spectrometry was examined. The methodology was validated and used to investigate the effect of hematocrit on assay bias using partial and whole spot extractions from accurately dispensed blood samples. Results: The completed analysis of a DBS sample was accomplished within 2 to 3 min using the online DBS-SPE platform. Hematocrit related bias was observed (>15%) for the partial DBS extractions, but not when the whole DBS was eluted. Conclusion: Results demonstrate successful implementation of automated online DBS-SPE high-resolution accurate mass spectrometry analysis and the remediation of hematocrit bias using a capillary micro dispenser for accurate spotting of blood samples.