Spent brewer's yeast (SBY) application as a new source of peptides depends on the development of proper downstream processes. This work aimed to separate yeast hydrolysate peptides from sugars and ribonucleic acids (RNA) by a 3-step designed fractionation process using ceramic ultra and nanofiltration following two sequences: (1) 50, 8 and 1 kDa and (2) 15, 8 and 1 kDa molecular weight cut-off (MWCO) membranes. In step one, 15 kDa MWCO membrane retained more components and increased performance of subsequent steps, when compared to 50 kDa. Peptide composition of the initial hydrolysate was gradually changed until 90% of the peptides smaller than 1 kg mol−1 were found in the final permeate. Fractionation increased peptide purity regarding RNA and total sugars up to 1.7 and 2.7-fold, respectively. Innovative protein-rich ingredients from SBY residue with an RNA content as low as 1.4% were produced by membrane separation technology. High quality ingredients presented different peptide profiles and great potential in food and pharmaceutical industries applications.