The increasing prevalence of multi-drug resistance within clinically relevant bacterial species is threatening the efficacy of our existing antibiotic classes. Compounding the issue is our current lack an effective antibiotic drug discovery platform. One of the main issues hindering the development of novel antibacterials is that there is a lack of knowledge regarding the physico-chemical properties required of compounds to accumulate within the bacterial cytoplasm. In this study, I developed an LC/MS based method which will allowed the screening of chemically diverse small molecules for accumulation within the cytoplasm of E. coli. This method could be used in future small molecule screens, from which we may attempt to identify structure activity relationships associated with bacterial penetration and efflux avoidance. This study also revisited the role of membrane carriers in the entry of antibiotics within bacteria. To assess this, I designed a screen which would allow the identification of membrane transporters which play a putative role in drug uptake, using a library of S. aureus strains containing transposon disruptions in non-essential membrane transporter genes. Using this screen, 30 carriers were identified to play a putative role in the uptake of 9 antibiotics from different drug classes. Further characterisation using genetic complementation, competition studies, drug accumulation assays and the generation of strains containing disruptions in multiple genes associated with drug uptake then confirmed the role of membrane carriers in the uptake of gentamicin, ciprofloxacin, chloramphenicol, tetracycline, fosfomycin and D-cycloserine. The results of this study show that membrane transporters play a previously unrealised role in the entry of antibiotics within S. aureus; this challenges the idea that drug entry occurs predominately via lipoidal diffusion, within bacterial cells.