Rapid determination of L-citrulline and L-arginine, physiologically important amino acids, is a beneficial technique from the scientific and medical viewpoints. In this study, enzymatic assays for L-citrulline and L-arginine were established and evaluated. L-Citrulline assay was constructed by coupling argininosuccinate synthetase to a pyrophosphate detection system, in which pyruvate phosphate dikinase was employed, so that the citrulline-dependent production of pyrophosphate could be determined. Furthermore, the L-arginine assay was developed by coupling arginine deiminase to the L-citrulline assay. Both assays exhibited high selectivity to L-citrulline and L-arginine without any significant reactivity to other proteinaceous amino acids. These assays were also resistant to various contaminants that interfered with the conventional L-citrulline and L-arginine assays. The high accuracy of these assays was demonstrated by measurements in the presence of human plasma. Because these assays can be conducted under the neutral pH without terminating the reaction progress, they allow not only measurements in static analyte solutions, but also real-time monitoring of L-citrulline and L-arginine synthesis in the reaction mixture. The features of these assays also demonstrated that the pyrophosphate detection system served as a useful platform to develop selective and robust enzymatic assays by being coupled to a pyrophosphate-producing enzyme. (c) 2014 Elsevier Inc. All rights reserved.