Introduction: The Quantase phenylalanine assay, an enzymatic/colorimetric method for the determination of phenylalanine in blood, was evaluated in combination with the Millipore Multiscreen Assay System (MMAS) for use as a microtiter plate assay system in neonatal PKU screening. Methods: A 3 16- inch disc was punched from the filter paper blood specimen and extracted in trichloroacetic acid. The MMAS was used for the transfer of the extracts to the microtiter plate wells. The Quantase phenylalanine assay was applied to these wells and the results read colorimetrically. Results: The Quantase assay was linear to a phenylalanine concentration at least 1.2 mmol/1. The sensitivity was at a phenylalanine level of 0.04 mmol/l. Within-run imprecision was 5–10% and between-run imprecision 8–9%. The mean measured phenylalanine level in 33627 normal newborn blood specimens was 0.77 ± 0.025 mmol/1. Of 13 samples classified as suspicious or positive for PKU for the Quantase method, seven were confirmed by the Guthrie bacterial assay. No sample positive by Guthrie testing was normal on Quantase assay. The MMAS facilitated this microtiter plate assay, providing adequate transfer of extract. Each MMAS plate could be reused up to three times with washing. Discussion: The Quantase phenylalanine assay in combination with the Millipore Multiscreen Assay System offers a reliable system for PKU Screening in the newborn.