The possibility to combine proteomics workflows to classical experimental approaches is continuously opening new insights in all branches of biological sciences. Deoxynivalenol (vomitoxin, DON) is a secondary metabolite produced by Fusarium spp. fungi and it is one of the most recurrent mycotoxins worldwide. DON is known to inhibit protein synthesis and as such interact with the cell models in multiple and complex ways whose comprehension enormously benefit of the system toxicology approach. For the purpose of this study epidermoid squamous cell carcinoma cells A431 were incubated with DON for 24h and toxin dependent variation of the proteome profile was obtained with a LC-MS/MS approach using a reversed phase nanoLC-System (Ultimate 3000RSLC Thermo Fisher Scientific, Austria) hyphenated to a High Resolution Orbitrap Mass Spectrometer (Thermo Fisher Scientific™ Q Exactive™ Classic). DON significantly down-regulated ribosomal proteins, as well as mitochondrial respiratory chain elements and transport proteins (TOMM22; TOMM40; TOMM70A). In line with the loss of mitochondrial function, altered metabolic capability was observed, with particular impairment of lipid synthesis cascade. Effect of the mycotoxin on cell membrane was verified by confocal microscopy (morphology) and by membrane fluidity measures (biophysical properties).