Venomous marine gastropods of genus Conus utilise one of the most sophisticated envenomation strategies known, with a single venom comprising of complex mixture of peptides evolved to target neuronal receptors, ion channels and transporters with high affinity and specificity. Arguably 800 species of cone snails exist throughout tropical and subtropical waters, with each species typically expressing ~100 major peptides and 1000–8000 related variants. Despite of this vast diversity, <0.1% of conotoxins are pharmacologically characterized, with the majority of characterized peptides being derived from fish hunting cone snails. However, traditional low throughput approaches and limited venom availability hindered the discovery of low expressing novel conotoxins. To overcome this limitation and accelerate the discovery and characterization of novel conotoxin classes from fish hunting cone snails of the clade Pinoconus, we have established a venom peptide discovery pipeline by integrating high-resolution venom transcriptomics with state-of-the-art proteomics. In this study we have optimised short read transcriptome assembly and analysis methods to reveal the full complexity of the venom duct transcriptomes. Proteomic visualisation methods were then used to unravel the venom distribution patterns across the venom duct to gain an understanding venom peptide regulation in the Pinoconus clade. The results of this study allowed the first detailed deciphering of multiplexed conotoxin diversification, across the venom duct, among individuals of the same species and across different species of Pionoconus clade. The venom duct compartmentalization is more complex in cone snails of the Pionoconus clade with different peptides with potentially similar pharmacologies are used exclusively for predation and defense and produced in distinct areas of the venom duct. Collectively this study help advancing our understanding of the multi-layered venom variability, complex biochemical innovations used for predation and defense while revealing a library of novel conotoxin families with potential as research tools.