Oligopeptides are a highly diverse nitrogen source for Saccharomyces cerevisiae during alcoholic fermentation. However, they are seldomly considered by the fermentation industry due to difficulties associated with their qualitative and quantitative measurement in natural matrices. Due to the limited studies that have been performed on oligopeptide transport systems in yeast, specifically the physiology of oligopeptide uptake during fermentation in a peptide rich environment, we developed a high-throughput UHPLC-IMS-HMRS methodology to follow the relative consumption of peptides derived from a bovine serum albumin hydrolysate during fermentation. This methodology was applied to a set of fermentations performed with haploid, CRISPR-Cas9-engineered peptide transporter knock-out strains derived from the S. cerevisiae wine strain EC1118® to study oligopeptide transporters from the Opt and Fot families. Results show that Opt2 can import peptides containing three to -at least seven amino acid residues, which is a broader peptide length specificity than previously reported, while Opt1 was shown not to be functional in these strains. It was also reported that Fot1, Fot2 and Fot3, previously referred to as di-tripeptide transporters in S. cerevisiae wine strains, are also able to import tetrapeptides. Yeast cells preferred the activity of Fot, as Opt2 activity was observed after most di-tetrapeptides were depleted from the media. Additionally, we provide further evidence of the importance of peptides as a nitrogen source for yeast and their consequent positive impact on fermentation kinetics. Fot and Opt2 assured completion of the fermentation process without necessarily requiring ammonia or amino acids.