Determining the diets of threatened Spectacled Eiders (Somateria fischeri (Brandt, 1847)) in relation to life-history stages will provide information to help identify and characterize their critical habitats. Quantitative fatty acid signature analysis (QFASA) is a novel tool that estimates the proportion of diet items in consumers from their fat depots. We conducted feeding experiments to validate the use of QFASA to estimate the mixed diets of captive female Spectacled Eiders using egg yolk fatty acids (FA) collected in 2008 and 2009. Calibration coefficients (CCs) for individual FA were developed to account for FA modification (due to eider lipid metabolism) from diets of eiders into egg yolk. We also compared the FA profiles between fertile and infertile eggs. Egg yolk FA profiles did not differ significantly between infertile and fertile eggs collected in either year. Using the CCs developed from eggs collected in 2008, QFASA closely estimated the 2009 diet composition of eiders. We conclude that using infertile eggs has the potential to provide a noninvasive method to elucidate diets of breeding female Spectacled Eiders and possibly other avian species, and to provide insight into understanding the sources (i.e., marine wintering or freshwater breeding habitat) and timing (i.e., prebreeding or breeding) of nutrient acquisition during reproduction.