Antibiotic resistance is a fast-growing crisis worldwide and has been estimated to cause 10 million deaths by the year 2050, which is more than the present number of deaths caused by cancer and road accidents combined. Recently, WHO has published a priority list of pathogens against which new antibiotics are urgently needed. In this list, Pseudomonas aeruginosa has been listed among the Priority-1 (critical) pathogens. Among the various resistance mechanisms used by P. aeruginosa, the use of Resistance-Nodulation-Division (RND) efflux pumps is an intrinsic resistance mechanism. RND efflux pumps are the tripartite complex consisting of outer membrane protein (OMP), membrane-fusion protein (MFP), and RND antiporter. Among 12 well-characterized RND efflux pumps found in the genome of P. aeruginosa, the MexXY efflux system has been reported to be overexpressed in the clinical isolates, especially isolates obtained from Cystic Fibrosis (CF) patients. Overexpression of the MexXY efflux pump leads to reduced susceptibility against aminoglycosides, a routinely used antibiotic for the treatment of CF patients. Understanding the process of antibiotic resistance through RND efflux pumps requires the knowledge of protein-protein interaction among RND complexes and their mechanism and determinants of substrate selectivity. This study is focused on understanding the role of RND efflux systems in multidrug resistance among CF isolates and determining the substrate determinants of an RND complex. Determinants of substrate selectivity were studied using MexXY-OprA/OprM efflux system as a model due to its clinical significance among CF isolates of P. aeruginosa, and its ability to form a functional complex with two different OMPs. Since OprA is only found in P. aeruginosa PA7 strain (and PA7-type genomes), we also compared the genome of PA7 with PAO1 (type-strain) to find any other clinically-relevant antibiotic resistance and virulence genes. Our study shows MexXY to be the most overexpressed RND system among the CF isolates of P. aeruginosa. We also showed that MexXY complexes with OprA and OprM have some differential substrates (carbenicillin and piperacillin). Using comparative genomics and proteomics approaches, we show that the PA7 genome harbors additional aminoglycoside modifying enzymes that confer resistance to several aminoglycosides. So, together, this study adds to the knowledge of RND efflux pump-mediated antibiotic resistance in terms of substrates selectivity and interplay between OMPs and RND antiporter. This knowledge of substrate determinants in RND efflux systems will aid in designing therapeutic options to treat multidrug-resistant infections.