Trace components present in biodiesel have been shown to contribute to the precipitation of solids at temperatures above the cloud point. These precipitates represent an operability problem for use of biodiesel in cold climates. Separation methods for analysis of trace lipid impurities, such as gas and liquid chromatography, have been problematic, and chromatographic signals for these compounds are often eclipsed by the signals for the fatty acid methyl esters (FAMEs), the major components of biodiesel. The method described herein has been developed as a rapid procedure for analyzing non-FAME lipid biodiesel components, which have been postulated to contribute to cold-weather operability problems. Representative standards of the non-FAME lipids present in biodiesel were analyzed with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) using pure and mixed matrix systems. An optimized ternary matrix system consisting of dithranol, 2,5-dihydroxybenzoic acid, and alpha-cyano-4-hydroxycinnamic acid doped with sodium iodide was developed for analysis of the widest range of trace components possible by capitalizing on the positives of each matrix in ionizing compounds with differing functional groups. Mixtures of matrix compounds produced smaller, more homogeneous crystals, which resulted in increased reproducibility and sensitivity. This increase in reproducibility allowed quantitative relationships to be established with standards and between fuel samples. Spectral peak identification was based on molecular weight and tandem mass spectrometry collision-induced dissociation. Two palm oil-derived biodiesels, one of which was distilled, were analyzed to determine their non-FAME components and to quantitatively compare the number and relative concentration of trace species detected. Trace lipid species in precipitates from canola-oil-derived biodiesel were also isolated via refrigerated centrifugation, followed by analysis with the ternary matrix.