Among the recognized classes of natural products, ribosomally synthesized and post-translationally modified peptides (RiPPs) represent an appealing group of molecules for advancing therapeutic development due to their biologically active characteristics (e.g., antimicrobial [1], antiviral [2], etc.). In contrast to bacteria [1], there has been limited research so far on RiPPs produced by fungi [3]. Despite the capability of genome mining tools to predict precursor peptide sequences for RiPPs, the extensive and diverse post-translational modifications arising during their maturation make it nearly impossible to anticipate their final structure. To establish a working protocol, initial experiments were conducted by investigating well-known fungal RiPPs from the Aspergillus flavus fungus. Various strains, including wildtype, knock-out, and overexpression strains of these gene clusters, were examined. Standard proteomics methods adequately identify the precursor peptide and related modifying enzymes within the gene cluster, but they fall short in identifying the fully mature peptide. Instead, comprehensive untargeted metabolomics techniques are employed to identify features displaying altered expression, ideally encompassing the desired mature RiPP. Metabolomics analyses apply both reversed-phase (RP) and hydrophilic interaction chromatography (HILIC), in positive and negative ionization modes, to cover the vast chemical space occupied by RiPPs. Rather than pinpointing individual metabolites, the approach subjects extracted features to a feature-based molecular network search, resulting in a molecular metabolite network. Given that the amino acid sequence and hence the mass of the RiPP precursor are known, it is envisioned that by analyzing the feature network containing the RiPP precursor, it becomes feasible to track the RiPP intermediates up to the fully mature form. This methodology is planned for adaptation to newly identified potential fungal RiPP candidates. [1] P. G. Arnison et al., Ribosomally synthesized and post-translationally modified peptide natural products: Overview and recommendations for a universal nomenclature. Nat. Prod. Rep. 2013, 30(1):108-160. doi:10.1039/c2np20085f [2] Y. Fu et al., Antiviral activities and applications of ribosomally synthesized and post-translationally modified peptides (RiPPs). Cellular and Molecular Life Sciences. 2021, 78(8):3921-3940. doi:10.1007/s00018-021-03759-0 [3] S. C. Kessler et al., Out for a RiPP: Challenges and advances in genome mining of ribosomal peptides from fungi. Nat. Prod. Rep. 2022, 39(2):222-230. doi:10.1039/d1np00048a