The acquired pellicle is a tenacious organic layer covering the surface of teeth, protecting the underlying dental hard tissues. Lipids account for about one quarter of the pellicle's dry weight and are assumed to be of considerable importance for their protective properties. Nevertheless, only preliminary information is available about the nature of lipids in the pellicle. Gas chromatography coupled with electron impact ionization mass spectrometry was used to establish a convenient analytical protocol in order to obtain a qualitative and quantitative characterization of a wide range of FAs (C ₁₂-C ₂₂). In situ biofilm formation was performed on bovine enamel slabs mounted on individual splints carried by 10 subjects. A modified Folch extraction procedure was adopted to extract the lipids from the detached pellicle, followed by transesterification to fatty acid methyl esters using methanol and concentrated hydrochloric acid. Tridecanoic and nonadecanoic acid were used as internal standards suitable and reliable for robust, precise and accurate measurements. The present study demonstrates, for the first time, a procedure based on a combination of innovative specimen generation and convenient sample preparation with sensitive GC-MS analysis for the determination of the fatty acid profile of the initial oral biofilm.