Abstract Background Cefiderocol (CFDC) is a siderophore cephalosporin with broad activity against Gram-negative bacteria. Discrepancies in susceptibility testing for CFDC have been reported. In this study susceptibility testing was performed using broth and agar medium and disks from various manufacturers to evaluate the reproducibility across the different sources and determine the relationship between MIC and inhibition zone. Methods 12 Enterobacterales, 12 P. aeruginosa, 12 A. baumannii with in vivo efficacy results in the murine thigh infection model under humanized PK of CFDC were evaluated. MICs were determined (10 replicates per media per isolate per day) using broth microdilution with iron-depleted cation-adjusted Mueller-Hinton broth (ID-CAMHB) prepared from BD BBL, BD Difco, Oxoid and Merck. The disk zone was determined (3 replicates per media/disk per isolate per day) with CFDC disks from Hardy and Liofilchem and Mueller Hinton Agar from BBL and bioMerieux. Testing was conducted for 3 days using the same inoculum for MIC and disk diffusion studies on each day. Susceptibility was interpreted according to 2023 CLSI breakpoints. Results Good reproducibility of MIC was observed for the ID-CAMHB from each manufacturer for all isolates, except for 2 A. baumannii due to heavy trailing and skip. MIC variation was observed between ID-CAMHB from each manufacturer. Best categorical agreement with in vivo efficacy was observed for the MIC obtained with ID-CAMHB from BD BBL. Good reproducibility of disk inhibition zones were obtained for Enterobacterales and P. aeruginosa irrespective of the manufacturer of disks and agar medium. A. baumannii isolates with elevated MIC values frequently showed colonies in the inhibition zones, with more colonies appearing on BBL agar. This phenomenon was not reproducible, so larger variation of inner inhibition zones were observed with these isolates. Conclusion MIC determinations were reproducible with each ID-CAMHB medium, but variability was observed across the different ID-CAHMB; MICs obtained with BD BBL ID-CAMHB showed best correlation with in vivo data. Good categorical agreement was observed for MIC in BD BBL and BD Difco medium and disk zone, despite variable inhibition zones observed for A. baumannii. Disclosures Miki Takemura, n/a, Shionogi & Co., Ltd.: Stocks/Bonds Mizue Saiki, Ph.D., Shionogi & Co., Ltd.: employee Hidenori Yamashiro, Shionogi & Co., Ltd.: employee Naomi Anan, MSc, Shionogi & Co., Ltd.: employee Boudewijn L. DeJonge, PhD, Shionogi Inc.: Employee Christine M. Slover, PharmD, Shionogi,INC: Employee Christopher M. Longshaw, PhD, Shionogi BV: Employee Anne Henriksen, PhD, Shionogi: Employee Yoshinori Yamano, PhD, Shionogi HQ: Employee