High-resolution mass spectrometry based non-targeted screening has a huge potential for applications in environmental sciences, engineering and regulation. However, it produces large datasets for which full appropriate processing is a real challenge; the development of processing software is the last building-block to enable large-scale use of this approach. A new software application, SPIX, has been developed to extract relevant information from high-resolution mass spectral datasets. Dealing with intrinsic sample variability and reducing operator subjectivity, it opens up opportunities and promising prospects in many areas of analytical chemistry. SPIX has shown its ability to reveal relevant modifications between two series of large datasets, allowing, for instance, the study of the consequences of a given event on a complex substrate. Most of all – and it is to our knowledge the only software currently available allowing this – it can reveal and monitor any kind of reaction in all types of mixture.