iBench is an open-source tool that provides enhanced validation of Mass Spectrometry identification methods. In this updated version, iBench 2.0 takes high confidence peptide identifications from previously measured MS data and embeds the sequences in an in silico-only proteome as spliced or non-spliced peptides. The MS data can then be reanalyzed with the modified proteome, thereby representing a (pseudo) ground truth dataset, to enable benchmarking of an identification method. In particular, precision-recall curves are generated, comparing the fraction of PSMs identified which are correct and the fraction of all PSMs assigned by a method. Reference to this dataset: Soh WT, Roetschke HP, Cormican JA, Teo BF, Chiam NC, Raabe M, Pflanz R, Henneberg F, Becker S, Chari A, Liu H, Urlaub H, Liepe J, Mishto M. Degradation of proteins by human 20S proteasomes sheds light on the interplay between peptide hydrolysis and peptide splicing. Nat. Comm., accepted.