Optimization of the method for analysis of free fatty acids (FFAs) in biological samples by MALDI-MS with the use of Langmuir technology at the stage of sample application on the target is proposed. Removal of the aqueous-organic drop from the target spot and automatic spectra registration during mass spectrometric analysis are proposed for the previously developed method for FFAs profiling in biological samples, that includes the following stages: application of an aqueous drop, containing barium salt and DHB matrix, to the center of a MALDI target well; double application of a hexane extract from a biological sample, containing FFAs; destruction of a monolayer dried on the target well with 90 % aq. acetonitrile and MALDI-MS analysis. The optimized approach was tested in a toxicological experiment and revealed significant ( p < 0.05) changes in the relative concentrations of a number of FFAs in the blood plasma of the control and experimental groups of rats.