Several studies have shown the ability of yeast to consume peptides as a nitrogen source in single-peptide containing media. However, a suitable and cost-effective methodology to study the utilization of peptides by yeast and other microorganisms in a complex peptide mixture has yet to be put forward. This article addresses this issue by presenting a screening methodology for tracking the consumption of peptides by yeast during alcoholic fermentation. As a peptide source, the methodology makes use of an in-house prepared peptide-mapped bovine serum albumin (BSA) proteolytic digest, which was applied to a synthetic grape must. The peptide uptake was analyzed using high-throughput ultra-high-pressure liquid chromatography coupled to data-independent acquisition-based ion mobility separation-enabled high-resolution mass spectrometry (UPLC-DIA-IMS-HRMS) analysis. The relative changes of abundance of 123 di- to hexapeptides were monitored and reported during fermentations with three commercial wine strains, demonstrating different uptake kinetics for individual peptides. Using the same peptide-mapped BSA hydrolysate, the applicability of an untargeted workflow was additionally assessed for peptide profiling in unelucidated matrixes. The comparison of the results from peptide mapping and untargeted analysis experiments highlighted the ability of untargeted analysis to consistently identify small molecular weight peptides on the length and amino acid composition. The proposed method, in combination with other analytical techniques, such as gene or protein expression analysis, can be a useful tool for different metabolic studies related to the consumption of complex nitrogen sources by yeast or other microorganisms.