There are many significant obstacles to producing high quality metabolomics datasets based on mass spectrometry. The identification of unknowns, including the determination of whether they are of biological or artifactual origin, and reduction of sample-to-sample variances, including suppression, are just a few of the issues that are generally considered intractable, and therefore often ignored. The IROA protocol is one in which all biological peaks are isotopically labeled in a manner that allows for the correction of both of these problems. The protocol induces a distinct isotopic patterning that makes it easy to identify by an automated software approach. The protocol and software combine to make it easier to produce high quality metabolomics experiments. The use of the IROA protocol in bacterial, fungal, and small model organisms such as C. elegans was demonstrated.