A simple, robust and reliable method has been developed for the quantification of fatty acids in used cooking oil (UCO) by gas chromatography–mass spectrometry (GC–MS). Four steps involved in this study are: the preparation of UCO for quantification analysis, development of calibration curve, determination of fatty acids composition and the ageing studies. The developed method has been validated with known composition of the UCO and biodiesel and the error has been found to be within ±0.7%. This method can be used for the determination of composition of fatty acids and fatty acid methyl esters (FAME) in oil and biodiesel products.