Background: Neonatal sepsis is one of the leading causes of neonatal morbidity and mortality. Fast and accurate antibiotic susceptibility testing is very important for early diagnosis and treatment with appropriate antimicrobial agents. The European Committee of Antimicrobial Susceptibility Testing provides rapid antimicrobial susceptibility testing (RAST), based on the disk diffusion method, after 4, 6, and 8 hours of incubation. This susceptibility testing directly from positive blood culture bottles was prospectively evaluated against the golden standard CLSI disc-diffusion method. Methods: A cross-sectional diagnostic study was conducted at the NICU in Ain Shams University Hospitals from October 2022 until September 2023. Overall, 115 positive blood cultures for Gram positive and negative bacteria were isolated. Antibiotic discs used were Imipenem, Ceftazidime, Trimethoprim-sulphamethoxazole, Piperacillin/tazobactam, Cefotaxime, Tobramycin, Gentamycin, Cefoxitin, and Clindamycin. The results were assessed using the RAST breakpoints after 4,6 and 8 hours against disc diffusion method of CLSI. Categorical agreement of RAST with disc diffusion method for these antibiotics was evaluated. Results: Matching the results of the susceptibility of all organisms to different antibiotics between the conventional golden standard method and EUCAST RAST method, Categorical agreement was 76.8%, 92%, and 97.2% at 4, 6, and 8 hours respectively. Conclusion: RAST is a promising method for rapid antimicrobial susceptibility testing with a high rate of categorical agreement with the conventional disc diffusion method, particularly at 8-hour incubation. Thus, it promotes the appropriate use of antimicrobials, mitigates the emergence of antimicrobial resistance, and improves patient quality of care and outcome.