Abstract A colorimetric sulfo-phospho-vanillin (SPV) method was used to validate a high throughput method for total lipids analysis in fresh and lyophilized oleaginous microorganisms. This method uses a small amount of biological material, does not require a lot of sample manipulation, can be automated, is reproducible and easy to implement. A 96-well microplate SPV assay allows one to determine quickly total lipids in lyophilized cells of oleaginous yeast and microalgae. The new assay method possesses many advantages compared to the others described in the literature: requires a small amount sample, less time (around 1 h) and less labor and does not require organic reagents like chloroform in the reaction.