As a promising source of biodiesel, Chlorella has drawn more and more attention of researcher, while rapid screening of enhanced lipid production becomes essential. Here we investigated lyophilized alga powders as the starting material and optimized Nile red method for quantitative measurement of neutral lipids in Chlorella with a high correlation coefficient (R-2 = 0.99) between gravimetric and spectrofluorimetric quantification. Using this method, Chlorella vulgaris were screened out of several Chlorella strains with the highest lipid content and orthogonal design experiments were performed to search for the significant factors affecting lipid accumulation. Our data implied that the sensitivity and versatility enable this method a useful tool in optimizing culture and accumulation condition of lipid production in alga and figured out several factors significantly influenced lipid production in C. vulgaris. (C) 2009 Elsevier Ltd. All rights reserved.