Sepsis, a life-threatening immune response to blood infections (bacteremia), has a ∼30% mortality rate and is the 10th leading cause of US hospital deaths. The typical bacterial loads in adult septic patients are ≤100 bacterial cells (colony forming units, CFU) per ml blood, while pediatric patients exhibit only ∼1000 CFU/ml. Due to the low numbers, bacteria must be propagated through ∼24-hours blood cultures to generate sufficient CFUs for diagnosis and further analyses. Herein, we demonstrate that, unlike other rapid post-blood culture antibiotic susceptibility tests (ASTs), our phenotypic approach can drastically accelerate ASTs for the most common sepsis-causing gram-negative pathogens by circumventing long blood culture-based amplification. For all blood isolates of multi-drug resistant pathogens investigated (Escherichia coli, Klebsiella pneumoniae, and Acinetobacter nosocomialis), effective antibiotic(s) were readily identified within the equivalent of 8 hours from initial blood draw using <0.5 mL of adult blood per antibiotic. These methods should drastically improve patient outcomes by significantly reducing time to actionable treatment information and reduce the incidence of antibiotic resistance. © 2018 International Society for Advancement of Cytometry.