A cell-based assay system for simultaneous quantification of the three amino acids, phenylalanine (Phe), methionine (Met), and leucine (Leu) in a single biological sample, was developed and applied in the multiplex diagnosis of three key metabolic diseases of newborn babies. The assay utilizes three Escherichia coli auxotrophs, which grow only in the presence of the corresponding target amino acids and which contain three different fluorescent reporter plasmids that produce distinguishable fluorescence signals (red, green, and cyan) in concert with cell growth. To mixtures of the three auxotrophs, immobilized on agarose gels arrayed on a well plate, is added a test sample. Following incubation, the concentrations of the three amino acids in the sample are simultaneously determined by measuring the intensities of three fluorescence signals that correspond to the reporter plasmids. The clinical utility of this assay system was demonstrated by employing it to identify metabolic diseases of newborn babies through the quantification of Phe, Met, and Leu in clinically derived dried blood spot specimens. The general strategy developed in this effort should be applicable to the design of new assay systems for the quantification of multiple amino acids derived from complex biological samples and, as such, to expand the utilization of cell based analytical systems that replace conventional, yet laborious methods currently in use.