The direct-transesterification method based on acetyl chloride and methanol was optimized to extract fatty acid methyl esters from Chlorella vulgaris by selection of a proper internal standard and optimization of extraction parameters. Glyceryl triheptadecanoate was proven to be the right internal standard for quantification of fatty acid methyl esters from microalgae. The optimized extraction parameters were more mildness, which were 80 degrees C for 45 min with 1/9 of chloride/methanol (v/v) and 80 mL/g of solvent/dried biomass. More importantly, the authors' optimized method was quite robust for either autotrophic or heterotrophic microalgae as the fatty acid methyl esters yield changed less than 5% with the varied parameters.