Abstract During the past two decades interest in linking soil microbial community composition and activity with ecosystem scale ﬁeld studies of nutrient cycling or plant community response to disturbances has grown. Despite its importance there are challenges in making this linkage. Foremost is the question of analytical feasibility. In general, microbiological community-level methodologies have not been readily adaptable to the large sample sizes necessary for ecosystem-scale research. As a result, it has been difﬁcult to generate compatible microbial and ecosystem data sets. Soil lipid analysis shows potential as a middle ground between simple biomass measures and molecular proﬁling. However, the two protocols that have most often been followed are either rapid but indiscriminate (total lipid analysis or fatty acid methyl ester analysis; FAME), or precise but time consuming (phospholipid fatty acid analysis; PLFA). In this paper we report results from a standardized soil used test a modiﬁed extraction method (the ‘hybrid’ method) developed to balance the speed of FAME and the precision of PLFA in order to increase sample throughput. In comparing the three methods, we ﬁnd that FAME and PLFA are qualitatively and quantitatively distinct. The FAME method yielded the highest fatty acid abundance, but also had high variance resulting in low precision. The PLFA method had precision, but low yield. The ‘hybrid’ method fell midway between FAME and PLFA for quantitative fatty acid yield. In addition, the hybrid extraction can be completed in a fraction of the time it takes for PLFA. The hybrid protocol appears to provide an optimal balance between effort and accuracy and therefore is a good choice for large-scale ecosystem studies.