BACKGROUND: Most first-tier newborn screening (NBS) biomarkers are evaluated by a 2-min flow injection analysis coupled to tandem mass spectrometry (FIA-MS/MS) assay. The absence of separation prior to MS/MS analysis can lead to false positives and inconclusive results due to interferences by nominal isobars and isomers. Therefore, many presumptive positive specimens require confirmation by a higher specificity second-tier assay employing separations, which require additional time and resources prior to patient follow-up. METHODS: A 3.2-mm punch was taken from dried blood spot (DBS) specimens and extracted using a solution containing isotopically labeled internal standards for quantification. Analyses were carried out in positive mode using a commercially available microfluidic capillary electrophoresis (CE) system coupled to a high-resolution mass spectrometer (HRMS). RESULTS: The CE-HRMS platform quantified 35 first- and second-tier biomarkers from a single injection in <2-min acquisition time, thus, successfully multiplexing first- and second-tier NBS for over 20 disorders in a single DBS punch. The CE-HRMS platform resolved problematic isobars and isomers that affect first-tier FIA-MS/MS assay specificity, while achieving similar quantitative results and assay linearity. CONCLUSIONS: Our CE-HRMS assay is capable of multiplexing first- and second-tier NBS biomarkers into a single assay with an acquisition time of <2 min. Such an assay would reduce the volume of false positives and inconclusive specimens flagged for second-tier screening.