Current methods for the analysis of fatty acid methyl esters (FAMEs) in rapeseed oil-based biodiesel refer to operationally defined measurands, which is a practical solution for routine analysis. In this paper, a new method for the SI-traceable quantification of selected FAMEs in biodiesel and its validation are described. This method has the potential to be a reference method for applications requiring structurally defined measurands and traceability to the SI as it allows direct comparisons to well-characterised calibrants through the use of isotopically labelled analogues of the analytes as well as establishing a full uncertainty budget. The method is based on gas chromatography-isotope dilution mass spectrometry. Its performance is demonstrated through its implementation and validation in two independent laboratories and is shown to provide reliable and traceable results for selected FAMEs in biodiesel test samples.