In this study, we investigated the physiological response of inorganic (Pi-PO4) deficiency; which has an increased effect on rhamnolipid production in P. aeruginosa, with respect to virulence and biofilm- related proteins. The proteins were obtained through extracellular and cytoplasmic proteomic analysis using non-rhamnolipid produced P. aeruginosa ΔrhlA strains (constructed for this study by deleting the rhlA gene from wild type PAO1, Nottingham Collection strain using the method of alleic exchange), and mono and di-rhamnolipid produced strain PAO1 wild type (Nottingham Collection strain), grown in the populously rhl-producing medium, the proteose- peptone ammonium salts (PPGAS).