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Abstract Background Aztreonam-avibactam (ATM-AVI) combination shows promising effectiveness on most carbapenemase-producing Gram-negatives, yet standardized antibiotic susceptibility testing (AST) methods for evaluating the combination in clinical laboratories is lacking. We aimed to evaluate different ATM-AVI AST approaches. Methods 96 characterized carbapenem-resistant clinical isolates belonging to 9 Enterobacterales (EB; n = 80) and P. aeruginosa (PA; n = 16) species, including 90 carbapenemase producers and 72 strains resistant to both CAZ-AVI and ATM, were tested. Paper disk elution (DE; Bio-Rad) and E-test gradient strips stacking (SS; bioMérieux) were performed for the ATM + CAZ-AVI combination. MIC Test Strip (MTS; Liofilchem) was evaluated for ATM-AVI MIC determination. Results were interpreted applying ATM clinical breakpoints of the EUCAST guidelines and compared to the broth microdilution method (Sensititre, Thermofisher). Results According to broth microdilution method, 93% of EB and 69% of PA were tested susceptible to ATM-AVI. The synergistic effect of ATM-AVI was of 95% for EB, but of only 17% for PA. The MTS method yielded higher categorical and essential agreement (CA/EA) rates for both EB (89%/91%) and PA (94%/94%) compared to SS, where the rates were 87%/83% for EB and 81%/81% for PA. MTS and SS yielded 2 and 3 major discrepancies, respectively, while 3 very major discrepancies each were observed for both methods. Concerning the DE method, CA reached 91% for EB and 81% for PA, but high number of very major discrepancies were observed for EB (n = 6; 8%) and for PA (n = 3; 19%). Conclusions The ATM-AVI association displayed excellent in vitro activity against highly resistant clinical Enterobacterales strains. MTS method offers accurate ATM-AVI AST results, while the SS method might serve as better alternative then DE method in assessing the efficacy of ATM + CAZ-AVI combination. However, further investigation is needed to confirm the methods' ability to detect ATM-AVI resistance.