Sepsis is a time-critical medical emergency, compounded by rising levels of antimicrobial resistance. The global AMR crisis has led to calls to reserve antibiotics for diagnostic test-justified use only. Unfortunately, culture-based methods rarely deliver a definitive result inside the critical clinical decision window, and we have little to offer in point of care testing. Even commonly used molecular diagnostic methods have limited use outside major laboratory medicine services. However, a recently introduced self-contained multiplex PCR array has promise. This blood culture identification (BCID) PCR panel reduced the time to BCID with consequent patient benefit in Western Australia field trials, but has a limited AMR detection repertoire. In order to address the challenge of expanding AMR diversity and the complexity of its underlying genotypes, we are working on rapid flow cytometry-assisted antimicrobial susceptibility (FAST) test methods that eliminate the secondary culture step. However, both mPCR blood culture identification array and the FAST method rely on a preliminary culture-based step. Trimming back the primary culture time requires a combination of molecular and cell biology systems to deliver speed, without reducing sensitivity and specificity. This has been achieved in blood cultures with a variant of fluorescent in situ hybridisation, which could be applied directly to uncultured blood. There are significant obstacles to the introduction of truly culture-independent diagnostic tests for sepsis in the clinical laboratory, not least the development of user-friendly test protocols, in vitro and clinical validation. We must embrace these challenges if we expect to continue our front line role against sepsis and AMR.