The problem of increasing rates of antibiotic resistance has become a global concern, particularly among multi drug resistant Gram-negative nosocomial pathogens. These organisms display non- susceptibility to the majority of routinely used antibiotics, causing infections which are more difficult to treat and increase the duration of patient recovery. Due to the plethora of resistance determinants and the molecular machinery which facilitates their dissemination, new strategies are required to investigate the mechanisms that confer antibiotic resistance. Proteomic techniques allow the global analysis of the expressed proteome, providing a more holistic view of the current physiological state of the bacterial cell. The techniques used in this investigation cover the separation, quantification and identification of proteins present in cellular extracts from resistant organisms. These included the use of 2-D electrophoresis, DIGE and LC-MS/MS mass spectrometry applied to multidrug resistant Escherichia coli, Klebsiella pneumoniae, Enterobacter c/oacae, Serratia marcescens and Acinetobacter baumannii. In summary, these investigations revealed that the Tol-Pal membrane protein system and susceptibilities to polymyxin antibiotics and biocides are altered upon acquisition of a resistance plasmid in E. coli. Furthermore, it revealed that non-carbapenemase-mediated carbapenem resistance in K. pneumoniae involved the loss of fimbriae proteins, the increased expression of OmpK26 and the resistance proteins ErnrA and APH(3"), in addition to OmpK35/36 porin loss. The upregulation of a multi drug efflux pump in E. c/oacae, A. baumannii and S. marcescens involved the differential regulation of many proteins, spanning a broad range of functional classes, including the MinCDE cell division inhibitors, iron acquisition proteins such as FepA and FhuA and proteins involved in biofilm and LPS formation such as PapC, LptD and GmhA. Overall this project has highlighted the complex and dynamic changes in protein expression upon acquisition of a resistance phenotype and the importance of using genetically related isolates when undertaking proteomic analyses. This work also emphasised the advantages of using proteomics for profiling the expression of resistance proteins, including the detection of specific enzymes, such as CTX-M ESBLs.