An automated method based on a hyphenated SPE-LC-MS/MS configuration has been optimized for the determination of essential amino acids (threonine, valine, methionine, leucine, isoleucine, lysine, tryptophan, and phenylalanine) in human serum, with the aim of discriminating between different states of coronary artery disease. Validation in terms of sensitivity (detection limits below 28.0ng on column) and precision (repeatability expressed as relative standard deviation below 6.0%) supports the suitability of the method for application to a cohort of 122 atherosclerosis patients confirmed by a catheterization test. The cohort was composed by 80 individuals diagnosed with stable angina and 42 patients who suffered from acute myocardial infarction (AMI). Both groups of individuals are differentiated by the occurrence of ischemia in AMI patients due to the formation of thrombi. The chemometric treatment of the data obtained by multivariate analysis of variance (MANOVA) allowed comparison between both groups of diagnosed patients. Therefore, amino acids whose serum levels were affected by ischemia have been identified. The contribution of risk factors such as obesity and hypercholesterolemia as well as the individuals' gender to the concentration of essential amino acids has also been studied.