Dried blood spots (DBS), collected for newborn screening programs in the United States, have been used to screen for congenital metabolic diseases in newborns for over 50 years. DBS provide an easy and inexpensive way to collect and store peripheral blood specimens and present an excellent resource for studies on the assessment of chemical exposures in newborns. In this study, a selective and sensitive method was developed for the analysis of iodide in DBS by high performance liquid chromatography electrospray tandem mass spectrometry. Accuracy, inter- and intraday precision, matrix effects, and detection limits of the method were determined. Further validation of the method was accomplished by concurrent analysis of whole blood and fortified blood spotted on a Whatman 903 filter card. A significant positive correlation was found between measured concentrations of iodide in venous whole blood and the same blood spotted as DBS. The method limit of detection was 0.15 ng/mL iodide. The method was further validated by the analysis of a whole blood sample certified for iodide levels (proficiency testing sample) by spotting on a filter card. Twenty DBS samples collected from newborns in New York State were analyzed to demonstrate the applicability of the method. The measured concentrations of iodide in whole blood of newborns from New York State ranged between <LOD and 16.4 ng/mL. The developed method is applicable for the analysis of DBS collected for epidemiological studies that investigate the importance of iodide on the health of newborns.