OBJECTIVES: Currently, newborns in Ontario, Canada, are screened for phenylketonuria (PKU) using the Guthrie method. Tandem mass spectrometry (MS/MS) can allow several other metabolic diseases to be screened for at birth. We estimated the costs, health benefits, and cost-effectiveness of using MS/MS to expand the newborn screening program in Ontario to include 20 additional metabolic diseases. METHODS: We constructed a decision-analytic model to estimate the incremental costs and life years of survival gained associated with screening by MS/MS versus the status quo (either not screening or screening with a different technology). Disease prevalence, test characteristics, treatment effectiveness, disease progression rates, and mortality were estimated from secondary sources and expert opinion. Costs were estimated primarily from the London Health Sciences Center Case Costing Initiative, the Ontario Health Insurance Plan Schedule of Benefits, and the Ontario Drug Benefits Plan formulary. Costs and health benefits were estimated for a cohort of babies born in Ontario in one year. RESULTS: Using MS/MS to screen for PKU has an incremental cost of $2,800,000 per life year (LY) gained. When each additional disease was evaluated independently, the cost of screening with MS/MS ranged from cost-saving to $158,000/LY gained. Using MS/MS to screen for PKU and 16 additional diseases costs $17,100/LY gained. CONCLUSIONS: Newborn screening reduces morbidity, mortality, and the social burden of irreversible effects of disease on the population. The severity and onset of disease symptoms can be prevented through early diagnosis and treatment. Our analysis suggests that screening for PKU and 16 additional diseases has an incremental cost effectiveness ratio below $50,000/LY gained. However, it is not cost effective to screen for all diseases that can be tested for using MS/MS.