Antimicrobial resistance (AMR) is a major threat to human health worldwide, and the rapid detection and quantification of resistance, combined with antimicrobial stewardship, are key interventions to combat the spread and emergence of AMR. Antimicrobial susceptibility testing (AST) systems are the collective set of diagnostic processes that facilitate the phenotypic and genotypic assessment of AMR and antibiotic susceptibility. Over the past 30 years, only a few high-throughput AST methods have been developed and widely implemented. By contrast, several studies have established proof of principle for various innovative AST methods, including both molecular-based and genome-based methods, which await clinical trials and regulatory review. In this Review, we discuss the current state of AST systems in the broadest technical, translational and implementation-related scope.