To compare truffles on protein level, samples of different truffle species (T. magnatum, T. indicum, T. melanosporum, T. aestivum and T. albidum Pico) were analysed by a LC-MS/MS based shotgun proteomics approach. First, different established bottom-up proteomics sample preparation approaches for in solution tryptic proteolysis were tested for a truffle sample. Then, a spectral library was generated by 2D LC-MS/MS and truffle samples were measured in data-independent acquisition (DIA) mode. We provide a comprehensive study on the proteomic differences between truffle species.