This protocol describes three methods for the quantitative determination of total fatty acid methyl esters (FAMEs) in marine sediments: One-Step Direct Transesterification Base-Catalyzed Saponification Acid-Catalyzed hydrolysis The one-step method combines extraction and transesterification into a single step, significantly reducing handling time and minimizing the risk of fatty acid degradation. Compared to the two conventional multi-step methods, the one-step protocol yields better recovery and improved repeatability, with no adverse effects on fatty acid profiles. The one-step procedure is an efficient, simple, rapid and economic alternative to multi-step methods for the routine analysis of total sedimentary fatty acids. Additionally, each protocol was tested with and without the use of BHT (butylated hydroxytoluene) as an antioxidant to assess its impact on lipid stability and recovery.