The lipid composition of microbial communities can indicate their response to changes in the surrounding environment induced by anthropogenic practices, chemical contamination or climatic conditions. A considerable number of analytical techniques exist for the examination of microbial lipids. This article reviews a selection of methods available for environmental samples as applied for lipid extraction, fractionation, derivatization and quantification. The discussion focuses on the origin of the standard methods, the different modified versions developed for investigation of microbial lipids, as well as the advantages and limitations of each. Current modifications to standard methods show a number of improvements for each of the different steps associated with analysis. The advantages and disadvantages of lipid analysis compared to other popular techniques are clarified. Accordingly, the preferential utilization of signature lipid biomarker analysis in current research is considered. It is clear from recent literature that this technique stays relevant – mainly for the variety of microbial properties that can be determined in a single analysis.