We have successfully optimized methods for extraction, methylation, and analysis of fatty acids for use on archaeological specimens. These analyses implement techniques that do not alter artifact integrity, facilitate analyte modification through transesterification to enhance analyte volatility for identification by direct injection GC–MS methods. Method development focused on the use of fatty acids found within the Great Basin of the USA, and has been designed using chemical stewardship to avoid environmental contamination and to protect the health of the analyst. In evaluating analysis methods, five traditional methylation techniques were examined only to prove inadequate for this study. By combining attributes of these methods with extraction goals, a modified single step extraction derivatization method was developed. Using this method we have demonstrated solid relationships between fatty acid ratios and plant/animal types. We have focused on method development and optimization for the detection of these fatty acids. In addition to optimization of instrumental variables we have compared various methylation methods to achieve optimal yields.