Progress in NGS technologies has opened up new opportunities for characterizing biodiversity, both for individual specimen identification and for environmental barcoding. Although the amount of data available to biologist is increasing, user-friendly tools to facilitate data analysis have yet to be developed. Our aim, with |SE|S|AM|E| Barcode, is to provide such support through a unified platform. The sequences are analysed through a pipeline that (i) processes NGS amplicon runs, filtering markers and samples, (ii) builds reference libraries and finally (iii) identifies (barcodes) the sequences in each amplicon from the reference library. We use a simulated data set for specimen identification and a recently published data set for environmental barcoding to validate the method. The results obtained are consistent with the expected characterizations (in silico and previously published, respectively). |SE|S|AM|E| Barcode and its documentation are freely available under the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported Licence for Windows and Linux from http://www1.montpellier.inra.fr/CBGP/NGS/. © 2012 Blackwell Publishing Ltd.