A simple and sensitive isocratic reversed-phase high-performance liquid chromatography (HPLC) method for simultaneous determination of retinol, alpha-tocopherol and six carotenoids in human plasma was described. Sample preparation of the earlier published method was further developed by addition of ultrapure water, which enabled aqueous layer to freeze facilitating phase separation without pipetting thus also improving precision of the method. Developed method appeared to be less laborious and time consuming compared to the traditional extraction methods, which require removal of organic layer by pipetting. The recoveries (absolute and relative) were between 80% and 103%. The intra-assay CVs were 1.1-4.0% (normal level) and 3.3-9.0% (low level). Inter-assay CVs were 5.3-8.8%. Reference method for all these analytes was not available, but a comparison with another published method was carried out. The results of the comparison matched satisfactorily. The method is used routinely in our laboratory in a large population-based study.