Updated project metadata. In this work we re-evaluate FASP and the related Multienzyme digestion (MED)-FASP method. We use different types of animal tissue and cultured cells, and test the performance of the method under various conditions. We analyze the protein-to-peptide conversion by assessing the yield of peptides, frequency of peptides with missed cleavage sites, and the reproducibility of FASP. We identify conditions allowing efficient protein processing with high peptide yields and demonstrate advantages of the two step digestion strategy over single step digestion with trypsin. In addition we show that FASP outperforms in solution cleavage strategies. Our results clearly show that the performance of the digestion varies between different types of samples. We show that MED-FASP in combination of the Total Protein Approach provide highly reproducible protein abundance values. The presented data can be used as guide for optimization of the sample processing