Ribosomally synthesized and post-translationally modified peptides (RiPPs) are an important class of natural products that include many antibiotics and a variety of other bioactive compounds. While recent breakthroughs in RiPP discovery raised the challenge of developing new algorithms for their analysis, peptidogenomic-based identification of RiPPs by combining genome/metagenome mining with analysis of tandem mass spectra remains an open problem. We present here MetaRiPPquest, a software tool for addressing this challenge that is compatible with large-scale screening platforms for natural product discovery. After searching millions of spectra in the Global Natural Products Social (GNPS) molecular networking infrastructure against just six genomic and metagenomic datasets, MetaRiPPquest identified 27 known and discovered 5 novel RiPP natural products. 1 spectra of natural products. However, to transform natural product discovery into a high-throughput technology and to fully realize the promise of the GNPS project, new algorithms are needed for natural products discovery 6-10 . Indeed, while spectra in the GNPS molecular network represent a gold mine for future chemical discoveries, their interpretation remains a bottleneck due to the large volume of data produced by modern mass spectrometers and unavailability of computational platforms for data processing.The efforts present herein focus on Ribosomally synthesized and Post-translationally modified Peptides (RiPPs), a rapidly expanding group of natural products with applications in pharmaceutical and food industries 11 . RiPPs are produced by RiPP Synthetases (RiPPS) through the Post Ribosomal Peptide Synthesis (PRPS) pathway 11 . RiPPs are initially synthesized as precursor peptides, encoded by RiPP structural genes. The RiPP structural genes are often quite short, making their annotation difficult 12 . A precursor peptide consists of a prefix leader peptide appended to a suffix core peptide. A leader peptide is important for recognition by the RiPP post-translational modification enzymes and for exporting the RiPP out of the cell. The core peptide is post-translationally modified by the RiPP biosynthetic machinery, proteolytically cleaved from the leader peptide to yield the mature RiPP, and exported out of the cell by transporters. The precursor peptide and the enzymes responsible for post-translational modifications (PTMs), proteolytic cleavage, and transportation usually appear in a contiguous biosynthetic gene cluster (BGC) of a RiPP within a microbial genome. The length of the microbial RiPP-encoding BGCs typically varies from 1,000 to 40,000 bp (average length 10,000 bp), larger than the current length of short reads generated by next generation sequencing (350bp), and making DNA assembly a critical part of any short read based RiPP discovery method.Genome mining refers to the informatics-based structural interpretation of a natural product BGC to infer information about the natural product itself. The discoveries of coelichelin in Streptomyces coelicolo...