PURPOSE: Accelerate Pheno provides rapid identification and antimicrobial susceptibility tests (ASTs) of pathogens that cause blood stream infections (BSIs). The study objective was to assess the accuracy of the Accelerate Pheno platform and its impact on antimicrobial modification in children with gram-negative BSIs. METHODS: A retrospective review was conducted of patients at a children's hospital with gram-negative BSIs from November 2018 to November 2019. Proportion of agreement between Accelerate Pheno and standard of care (SOC) was determined for organism identification (matrix-assisted laser desorption ionization time-of-flight mass spectrometry) and susceptibilities (MicroScan). Time from culture collection to Gram stain, identification and AST by the Accelerate Pheno method, and AST results by MicroScan were calculated. Antibiotic modifications and opportunities to optimize antimicrobial stewardship were recorded. FINDINGS: Of 115 BSIs from 90 patients, 90 monomicrobial gram-negative BSIs with an organism included on the Accelerate Pheno panel were found. Compared with SOC, the organism was correctly identified in 90 patients (100%). Overall, 5 of 732 ASTs (0.7%) reported susceptible by Accelerate Pheno were resistant by SOC, and 8 of 109 (7.3%) reported resistant by Accelerate Pheno were susceptible by SOC. On the basis of the Accelerate Pheno AST results, antibiotic spectrum was increased in 10 of 11 instances to correct organism-drug mismatch and narrowed in 16 of 33 instances. Median times from culture collection to reporting of Gram stain, Accelerate Pheno identification, Accelerate Pheno AST, and SOC AST were 12.6, 14.6, 19.9, and 60.6 h, respectively. Median time to optimal therapy was 21.8 h for infections with actionable AST data. IMPLICATIONS: Accelerate Pheno was accurate and decreased time to optimal therapy by almost 40 h for children with gram-negative BSIs. Antibiotic spectrum was increased in multiple instances, but opportunities to decrease spectrum were underused.