Abstract The validation of naturalness of different flavours (acetaldehyde-‘apple’ odour, acetoin —‘rancid butter’ odour, 2-methyl and 3-methylbutyric acids—‘rancid cheese’ odours) was made possible by developing a multi-component stable isotope picture for each compound. Flavour molecules analysed were of biotechnological or synthetic origin. The isotope results generated data that allowed for comparison of the isotopic abundance of known naturally derived or processed flavours with those of non-natural sources or processes. Our results confirmed that the biotechnological flavours had a natural origin and their isotope ratios were different from those of synthetic ones. Moreover, for a good discrimination at least two or three isotope analyses were necessary to prove the origin of flavours.