Expanded newborn screening uses tandem mass spectrometry (MS/MS) to identify patients affected with fatty acid oxidation defects by the presence of pathological acylcarnitine esters. A caveat to MS/MS assessment is that cut-off values for disease-specific acylcarnitines does not always clearly discriminate affected patients from carriers and healthy individuals. Diagnostic evaluation of screening-positive samples is required to confirm a metabolic deficiency. With MS/MS newborn screening becoming established in a growing number of countries, streamlined means for time- and -effective follow-on diagnostic evaluation is essential. Moreover, studies to evaluate the diagnostic accuracy of MS/MS newborn screening are needed for determination and adjustment of precise cut-off values for the disease-specific acylcarnitines. In the current study, we use the fatty acid oxidation disorder very-long-chain acyl-CoA dehydrogenase deficiency (VLCADD), the second most common fatty acid oxidation disorder detected by expanded newborn screening, to demonstrate accurate and fast diagnostic evaluation of the ACADVL gene utilizing DNA extracted from the newborn screening dried blood spot and high resolution melt (HRM) profiling. We also demonstrate that HRM is a very effective means to determine carrier frequency of prevalent ACADVL mutations in the general population. Based on estimates of the expected disease incidence, we discuss the diagnostic accuracy of MS/MS-based newborn screening to identify VLCADD in Denmark.