The aim of this study was to develop and validate a HPLC-MS/MS assay to determine the lutein concentration in plasma samples of human and SD rats. Organic solvent was used for lutein extraction. The extract was injected into a HPLC-MS/MS system. Reversed phase chromatography was performed on a C18 column in gradient mode. Lutein and internal standard (phenytoin sodium) were identified in atmospheric pressure chemical ionization mode using ion transitions of m/z 567.5>549.4 and 205.2>110.8, respectively. The lutein quantification assay was linear over concentrations ranging from 4 to 500 ng/mL. The lower limit of quantification was 4 ng/mL with satisfactory precision and accuracy. The assay presented acceptable intra and inter-batch precision (RSD%) and accuracy (RE%) <8.16% in SD rat plasma and <12.80% in human plasma. The extraction recovery ranged from 50.94 to 60.90% in SD rat plasma and 68.73% in human plasma. The matrix effect for lutein was acceptable and had minimal influence on the results. The method was then applied to determine the lutein concentrations in human plasma after a single oral dose of 20mg lutein. The method described is rapid, selective, sensitive and reproducible. This method can be used for both pharma-cokinetic studies and therapeutic drug monitoring purposes.