By measuring the potential glucose precursors entering and existing the liver, an estimate of the maximal rate of de novo gluconeogenesis can be made. Traditionally, measurements of gluconeogenic amino acids have been extracted from full amino acid profiles using conventional ion-exchange chromatography. These methods are labor intensive, costly procedures that do not focus on gluconeogenic amino acids. The present paper describes a method that provides an accurate whole blood gluconeogenic amino acid profile (intra-assay coefficients of variation from 0.8 to 1.1% and inter-assay coefficients of variation from 2.9 to 4.3%) using high-performance liquid chromatography with o-phthalaldehyde chemistry. This automated method is relatively fast (injection to injection time = 30 min), and linear (r2 > 0.996) for both standards and deproteinized whole blood. Furthermore, it is economical and capable of assessing gluconeogenic amino acids across a broad physiological range of concentrations using small sample volumes.