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This study aims to evaluate the accuracy of EUCAST rapid antimicrobial susceptibility testing (RAST) for Gram-negative bacteria directly from positive blood cultures, comparing it with short-term incubation (5-7 hours) and conventional broth microdilution methods. A total of 139 Gram-negative isolates were tested. RAST results were assessed at 4 h, 6 h against minimal inhibitory concentration results using the short-term incubation (5-7 h) method, while at 16-20 h, the RAST results were compared to conventional method. For those with interpretable results, CLSI M52 was used to define cutoffs for equivalence in antimicrobial susceptibility testing. Among all isolates, 80.6% (112/139) were successfully interpreted based on EUCAST RAST breakpoints, including Escherichia coli (81), Klebsiella pneumoniae complex (17), Pseudomonas aeruginosa (10) and Acinetobacter baumannii (4). The overall category agreements for all tested antibiotics were 98.9%, 99.5%, and 99.7% at 4, 6, and 16-20 hours, respectively, for E. coli, and 100% for K. pneumoniae, P. aeruginosa, and A. baumannii. The area of technical uncertainty rate significantly decreased over time, from 9.1% at 4 hours to 3.1% at 16-20 hours (p