Background Current in-silico based proteomics requires the prediction, validation and functional analysis of a modelled protein. The main drawback in this protocol is the lack of a singular protocol that utilizes a proper set of benchmarked open science tools to accurately predict a protein’s structure and function. Results The present study aims to utilize a series of openly available computational tools to formulate a complete pipeline capable of analyzing a novel protein’s amino acid sequence, reveal its phylogenetic relationships and conserved residues. The resultant data would then be utilized in the formulation of the protein’s tertiary structure followed by functional analysis through virtual screening. This novel protocol was benchmarked utilizing the Cystatin C protein obtained from Danio rerio and the AChE proteins of Homo sapiens and Rattus norvegicus. Conclusion The resultant pipeline utilizes freely available in-silico tools capable of complete protein analysis including a novel protein’s functional analysis. The methodology has been proven and the tools have been benchmarked as the most suited for complete trifecta protein analysis.