The dynamic predator–prey relations in the food web are vital for understanding the function and structure of ecosystems. Dietary estimation is a research hotspot of quantitative ecology, providing key insights into predator–prey relationships. One of the most promising approaches is quantitative fatty acid signature analysis (QFASA), which is the first generation of statistical tools to estimate the quantitative trophic predator–prey relationships by comparing the fatty acid (FA) signatures among predators and their prey. QFASA has been continuously widely applied, refined and extended since its introduction. This article reviewed the research progress of QFASA from development and application. QFASA reflects the long-term diet of predator, and provides the quantitative dietary composition of predator, but it is sensitive to the metabolism of predator. The calibration coefficients (CCs) and the FA subset are two crucial parameters to explain the metabolism of predators, but the incorrect construction or improper use of CCs and the FA subset may cause bias in dietary estimation. Further study and refinement of the QFASA approach is needed to identify recommendations for which CCs and subsets of FA work best for different taxa and systems.