A platform based on hydrophilic interaction chromatography in combination with Fourier transform mass spectrometry was developed in order to carry out metabonomics of Drosophila melanogaster strains. The method was able to detect approximately 230 metabolites, mainly in the positive ion mode, after checking to eliminate false positives caused by isotope peaks, adducts and fragment ions. Two wild-type strains, Canton S and Oregon R, were studied, plus two mutant strains, Maroon Like and Chocolate. In order to observe the differential expression of metabolites, liquid chromatography-mass spectrometry analyses of the different strains were compared using sieve 1.2 software to extract metabolic differences. The output from sieve was searched against a metabolite database using an Excel-based macro written in-house. Metabolic differences were observed between the wild-type strains, and also between both Chocolate and Maroon Like compared with Oregon R. It was established that a metabonomic approach could produce results leading to the generation of new hypotheses. In addition, the structure of a new class of lipid with a histidine head group, found in all of the strains of flies, but lower in Maroon Like, was elucidated.