Due to the rise of multidrug-resistant strains of Vibrio cholerae and the recent cholera outbreaks in African and Asian nations, it is imperative to identify novel therapeutic targets and possible vaccine candidates. In this regard, this work primarily aims to identify and characterize new antigenic molecules using comparative RNA sequencing data and label-free proteomics data, carried out with essential GTPase cgtA knockdown and wild-type strain of V. cholerae. We identified hitherto 51 characterized proteins from high-throughput RNA-sequencing and proteomics data. This work involved the assessment of their physicochemical characteristics, subcellular localization, solubility, structures, and functional annotations. In addition, the immunoinformatic and reverse vaccinology technique was used to find new vaccine targets with high antigenicity, low allergenicity, and low toxicity profiles. Among the 51 proteins, 24 were selected based on their immunogenic profiles to identify B/T-cell epitopes. In addition, 20 prospective therapeutic targets were identified using virulence predictions and related investigations. Furthermore, two proteins, UniProt ID- Q9KRD2 and Q9KU58, with molecular weight of 92kDa and 12kDa, respectively, were chosen for cloning and expression towards in vitro biochemical characterization based on their range of expression patterns, high antigenic, low allergenic, and low toxicity properties. In conclusion, we believe that this study will reveal new facets and avenues for drug discovery and put us a step forward toward novel therapeutic interventions against the deadly disease of cholera.