To assess the performance of the drop test for polymyxin B resistance detection among Enterobacterales and non-fermentative gram-negative rods resistant to carbapenems. Seven hundred and fifteen carbapenem-resistant isolates were tested: 628 Enterobacterales species and 87 non-fermentative gram-negative rods. For the polymyxin drop test, concentrations range from 0.25 to 8.0µg/mL. Broth microdilution, as gold standard, was performed using in-house-prepared panels and interpreted according to the CLSI guidelines. Results were interpreted in terms of categorical agreements and discrepancies. Accuracy for a drop of polymyxin B at 2.0, 4.0 and 8.0 was calculated as better cutoff for resistance determination. No very major error was observed among all isolates, and 95.5% of agreement was observed among Enterobacterales, particularly for Klebsiella pneumoniae. A higher accuracy (95.1%) was obtained when a single drop of polymyxin B at 4.0μg/mL was applied. Polymyxin drop test presented >95% of categorical agreement, without very major errors, for KPC-producing K.pneumoniae isolates. An accuracy of 95.1% was obtained with a single drop at 4.0μg/mL polymyxin B. Polymyxin B drop is an easy and feasible test and may allow a reduction on the turnaround time for polymyxin resistance detection and impacting on early implementation of accurate therapeutic interventions.