An experimental study was conducted to evaluate the druggability prioritization targets of E. coli in Gallus gallus. Total proteome of pathogen Vs host comparison was done using comparative genomics followed by protein docking of the target proteins of the pathogen and drug target identification and then calculation of drug prioritization parameters for therapeutic targets. 4,288 proteins of E. coli were compared with 24068 proteins of Gallus gallus using BLASTP analysis. 389 non-homologous proteins of E. coli were found, while 3,899 proteins were found homologous. These 389 non-homologous proteins were analyzed using BLAST against PDB and all targets from Drug Bank. Number of proteins found hits against PDB (sequences of proteins with known structure): 35 and against Drug Bank were 17. Thirty five proteins found with solved PDB structures were analyzed using CLASTLW and having an average alignment of above or minimal 30%. The thirty five proteins structures were also determined using PISA and the structures of the proteins were determined, of which 3 were monomers,11dimers, 2 trimers,tetramers, 2hexamers, 1octamer,1 nanome and 4 polymers,7 protein structures were not available with the database. The 35 proteins were loaded into Discovery studio version 4.1 for receptor ligand interaction studies with 32 ligands and ten proteins were found posed with ligand. The antibiogram study in phase II revealed the MIC values of all commonly used veterinary antimicrobials along with novel non-antimicrobial lead molecules (based on in silico studies) was performed and it was found that Cefpodoxime and Moxifloxacin showed the moderate in vitro activity, whereas the lead molecules have not been found with any susceptibilities at the given concentrations. The results of the study revealed further scope for development of potential drug targets for developing novel molecules against E. coli along with the status of susceptibility to different antibiotics.