A simple and rapid high performance liquid chromatographic (HPLC) method was developed to determine the major carotenoids in human serum. Lutein, b-carotene and lycopene were separated within 20 min using a C18 column and a mobile phase of methanol/methylene chloride (95:5, v/v) with sample solvent methanol/methylene chloride (45:55, v/v), detection at 476 nm and flow rate at 0.8 mL/min. A high recovery was achieved with this method, which amounted to 92, 90 and 87% for lutein, b-carotene and lycopene, respectively, while the detection limit was 5.0, 10.0 and 4.0 ng/mL. This method was applied to determine the major carotenoids in human serum samples collected from an elderly care center. A total of 62 samples were measured, and in most cases, bcarotene (17.5-1105.7 ng/mL) was present in the highest amount, followed by lutein (10.6-182.3 ng/mL) and lycopene (0-402 ng/mL).