This project provides tandem mass spectrometry datasets of an artificial mixture used to optimize the lysis and evaluate digestion protocols for a fast preparation of peptides for tandem mass spectrometry proteotyping. The microorganism mixture is composed of Bacillus subtilis subsp. spizizenii ATCC 6633, Escherichia coli BL21-Gold(DE3)pLysS AG and Saccharomyces cerevisiae S288C.