Microbial resistance to antibiotics is well established in the microbial world and remains a problem of significant clinical impact affecting all classes of anti-infective drugs. The collection of genes that encode antibiotic resistance mechanisms is the ‘antibiotic resistome' and understanding the chemical strategies that microbes use to overcome the toxic effect of antibiotics is essential to maintain our current arsenal of antibiotics and to guide the discovery and develop of new anti-infective agents. We have developed parallel approaches to collate the components of the resistome that include mining of microbial genomic data, literature searches of reported mechanisms, as well as systematic screens of environmental organisms for antibiotic resistance. Using chemical biology strategies, we are validating predicted resistance genes and probing molecular mechanisms of resistance for several classes of antibiotics. By developing an understanding of the biochemical logic of antibiotic resistance we are seeking to provide the requisite information for informed antibiotic use and new compound development.