Homoarginine (hArg) is a non-essential amino acid that was identified as a risk marker for cardiovascular disease. Several analytical methods have been described for the quantification of hArg in biological samples. The aim of this study was to compare a liquid chromatography-tandem mass spectrometric (LC-MS/MS) approach with a commercially available enzyme-linked immunosorbent assay (ELISA). Determination of hArg concentrations in ELISA calibration standards measured by both methods revealed a correlation coefficient r (2) of 0.99, for LC-MS/MS calibrators r (2) was 0.997. However, linear regression analysis between the two assays for hArg concentrations in human plasma samples revealed a correlation coefficient r (2) of 0.78. Plasma concentrations obtained from LC-MS/MS are on average 29 % higher than those by ELISA. We investigated the hArg-isobaric N (ε)-trimethyllysine as potential source for the higher observed values, but evaluation of mass spectra indicated that N (ε)-trimethyllysine did not interfere with hArg quantification in our LC-MS/MS method. Both quantification methods were applied to measure hArg in (1) a case-control study of acute coronary syndrome and (2) L-arginine:glycine amidinotransferase-deficient mice. Our LC-MS/MS and the commercially available ELISA assay are suitable for hArg measurement in human and mouse plasma, but different reference values for each method need to be considered.