Successful treatment of bacterial infections requires the timely administration of appropriate antimicrobial therapy. The failure to initiate the correct therapy in a timely fashion results in poor clinical outcomes, longer hospital stays, and higher medical costs. Current approaches to antibiotic susceptibility testing of cultured pathogens have key limitations ranging from long run times to dependence on prior knowledge of genetic mechanisms of resistance. We have developed a rapid antimicrobial susceptibility assay for Staphylococcus aureus based on bacterial cytological profiling (BCP), which uses quantitative fluorescence microscopy to measure antibiotic induced changes in cellular architecture. BCP discriminated between methicillin-susceptible (MSSA) and -resistant (MRSA) clinical isolates of S. aureus (n=71) within 1–2h with 100% accuracy. Similarly, BCP correctly distinguished daptomycin susceptible (DS) from daptomycin non-susceptible (DNS) S. aureus strains (n=20) within 30min. Among MRSA isolates, BCP further identified two classes of strains that differ in their susceptibility to specific combinations of beta-lactam antibiotics. BCP provides a rapid and flexible alternative to gene-based susceptibility testing methods for S. aureus, and should be readily adaptable to different antibiotics and bacterial species as new mechanisms of resistance or multidrug-resistant pathogens evolve and appear in mainstream clinical practice. •Bacterial cytological profiling identifies antibiotic resistant S. aureus.•BCP predicts best treatment options for multidrug resistant MRSA.•Resistant strains are correctly identified within 1h.•BCP does not require prior knowledge of resistance mechanism. There is a great need for rapid antimicrobial susceptibility testing (AST) as it can dramatically improve clinical outcome for bacterial infections. Most currently proposed ASTs are dependent on knowledge of known resistance genes or based solely on growth/lysis. We have developed a new diagnostic method for rapidly determining antibiotic susceptibility of Staphylococcus aureus using quantitative fluorescence microscopy to measure antibiotic induced changes in cellular architecture. Our test has the potential to change the way antibiotic susceptibility testing is done in the future and is readily adaptable to different antibiotics and bacterial species regardless of the mechanisms of resistance.