The aliphatic chains of fatty acids are the most prominent and potentially the highest value fuel precursor constituents of algal biomass, and thus accurately quantifying the algal biomass total fatty acid content is a prerequisite for comparing algal strains, growth conditions, and processes. Direct, acid-catalyzed transesterification of whole microalgal biomass is a simple, effective, strain-agnostic, and widely used method to determine the fatty acid content in whole algal biomass, with the precision needed to detect small but significant differences between strains. Such a direct transesterification procedure typically covers the following steps: first, solubilizing the lipids in the biomass matrix and then liberating the fatty acids to make these available for catalytic transesterification to fatty acid methyl esters (FAMEs), subsequent extraction into hexane, and then quantification by gas chromatography. The method we describe here requires less than 10 mg of biomass per sample and is considered high-throughput and highly accurate.