Background <p>Bloodstream infections (BSI) related mortality rates are increasing worldwide making it a medical emergency. This study has evaluated a new method for direct detection of pathogens and to perform direct antimicrobial susceptibility testing from the positive flagged blood culture. Early diagnosis and prompt treatment with appropriate antibiotics is the utmost need.</p> Method <p>A 30-minute protocol for pellet formation was developed using the positive blood culture bottle broth by triton X, SDS and saponin method for direct identification of pathogens. Clinical blood culture samples from patients, positive for Gram-negative bacteria, were included in the study (160 for direct identification and 250 for direct antimicrobial susceptibility testing). We compared results with routine method.</p> Results <p>The agreement of Triton X, SDS and saponin direct identification method compared to the conventional method was 96.2%, 91.8% and 90% respectively. A total of 960 pathogen and antimicrobial agent combinations were tested, 99.8% antimicrobial sensitivity testing results by direct method showed categorical agreement with the standard routine disc diffusion method.</p> Conclusion <p>Overall, the newer method of direct microbial identification and antibiotic sensitivity testing is both time and cost effective.</p>