A novel method for chemical fingerprinting of oil and petroleum products based on electrospray mass spectrometry (ESI-MS) and chemometrics has been used to identify and quantify surfactants in crude oil. The method is based on full-scan positive ESI-MS using a single quadrupole LC-MS instrument, however, with direct injection (no chromatographic separation) and without fragmentation of the molecules. The method was evaluated using two complex surfactant mixtures, Tween 80 and 85, which were added to a crude oil. Two dilution series were prepared with either Tween 80 or Tween 85 in the oil in concentrations ranging from 0.01 to 5.0%. In addition, samples of pure crude oil and pure surfactants were prepared. The samples were analyzed by positive ESI-MS. Similarities and differences between spectra were evaluated by means of principal component analysis (PCA). Projections to latent structures (PLS) was used for the multivariate calibration. With this method it was possible to identify and differentiate between the two surfactants at concentrations as low as 0.01%, although the method was optimized for a concentration range relevant for oil dispersants, which may be 100 times higher.