Non-targeted metabolomics is the hypothesis generating, global unbiased analysis of all the small-molecule metabolites present within a biological system, under a given set of conditions. It includes several common steps such as selection of biological samples, sample pre-treatment, analytical conditions set-up, acquiring data, data analysis by chemometrics, database search and biological interpretation. Non-targeted metabolomics offers the potential for a holistic approach in the area of biomedical research in order to improve disease diagnosis and to understand its pathological mechanisms. Various analytical methods have been developed based on nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS) coupled with different separation techniques. The key points in any analytical method development are the validation of every step to get a reliable and reproducible result and non-targeted metabolomics is not beyond this criteria, although analytical challenges are completely new and different to target methods. This review paper will describe the available validation strategies that are being used and as well will recommend some steps to consider during a non-targeted metabolomics analytical method development. Copyright © 2014 Elsevier B.V. All rights reserved.