Positive electrospray ionization mass spectrometry (ESI−MS) and multivariate regression (chemometrics) have been used for the identification and quantification of fatty acid methyl ester (FAME) in jet fuel in concentrations from 3 to 35 ppm. The jet fuel samples were injected directly and undiluted into the ion source. Each analysis takes less than 1 min to perform. Calibration series with rapeseed methyl ester (RME) and soybean methyl ester (SME) alone or in combination were used to create regression models with excellent prediction properties. An independent test set with known amounts of RME and SME was made several weeks later, and the regression model was used to predict the concentration of RME and SME with a root-mean-square error of prediction (RMSEP) of 2.6 and 1.2 ppm, respectively.